CN105925658A - Method for rapidly detecting joint toxicity of food additives (new red and sodium methyl parahydroxybenzoate) - Google Patents
Method for rapidly detecting joint toxicity of food additives (new red and sodium methyl parahydroxybenzoate) Download PDFInfo
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- CN105925658A CN105925658A CN201610244076.8A CN201610244076A CN105925658A CN 105925658 A CN105925658 A CN 105925658A CN 201610244076 A CN201610244076 A CN 201610244076A CN 105925658 A CN105925658 A CN 105925658A
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- reddest
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
Abstract
The invention discloses a method for rapidly detecting joint toxicity of food additives (new red and sodium methyl parahydroxybenzoate). According to the method, in accordance with the compound structures of the food additives (new red and sodium methyl parahydroxybenzoate) as well as experimental results of cytotoxic effects of the food additives, the following specific indicators for representing the joint toxicity of the new red and the sodium methyl parahydroxybenzoate are screened out: the total number of cells, the number of live cells, state of cell nucleuses, cell tubulin, mitochondrial membrane potential and lysosome PH; the six indicators are detected and analyzed by virtue of a high-content analysis system; and the joint toxicity is identified in accordance with an analysis result of the specific indicators. The method disclosed by the invention, which optimizes a detection process, has the advantages of being rapid, reliable, good in repeatability, strong in specificity and the like; the method offers a theoretical foundation to the right use of the food additives, namely the new red and the sodium methyl parahydroxybenzoate; and the method disclosed by the invention is of great significance for guaranteeing the right use of the food additives and for guaranteeing food safety.
Description
Technical field
The invention belongs to field of food safety, relating to one, quickly to detect food additive (the reddest, to hydroxyl
Yl benzoic acid methyl ester sodium) method of joint toxicity, it is specifically related to a kind of disposable quickly two kinds of food of detection
Additive: the reddest, Sodium Methyl Hydroxybenzoate adds the poisonous effect of generation simultaneously.
Background technology
Food additive is one of important component part in all processed food dispensings, and processing can be made to eat
Product have additional nutrients；Improve the aesthetic quality such as color, shape and inherent quality；Prevent food spoilage from becoming
Matter；Improve the processing conditions etc. of food.The use standard of the additive used at present is based on single thing
The toxicity of matter and formulate, and in food service industry, food additive is all used in combination, in real life
The mankind absorb numerous food every day, and a kind of food additive is safe in allowing range, and several
When kind uses simultaneously, the synergy of the form such as addition, collaborative, antagonism may be produced, also exist
Make the danger that the toxic action of one matter strengthens.Not yet there is at present the evaluation and test two kinds of standard and above
The method of food additive toxicity, therefore need badly foundation a kind of easy and simple to handle, can evaluate and test rapidly and accurately
The modernization detection method of food additive joint toxicity.
High intension analysis (high content analysis, HCA) is a kind of based on cell phenotype analysis
Efficient new medicament screen technology, it is possible to keep cellularity and fully functional on the premise of, detect simultaneously
By sieve sample form, growth, cycle, migration, apoptosis, metabolic pathway and signal conduction etc. to cell
The impact of aspect, it is achieved the multiple biological activity of compound and the early stage of toxicity, quick, high throughput testing.
Have simple to operate, rapidly and efficiently, in real time monitoring, high sensitivity, short time obtain and enrich data message
Etc. advantage.This technology is mainly used in the aspects such as single compound (novel drugs) screening at present, and to food
The method for quick of additive (the reddest, Sodium Methyl Hydroxybenzoate) joint toxicity do not have been reported that and
Open.
Summary of the invention
It is an object of the invention to for the deficiencies in the prior art, it is provided that a kind of discriminating food additive is (new
Red, Sodium Methyl Hydroxybenzoate) joint toxicity method, particularly a kind of disposable quickly detection is the reddest,
The method of Sodium Methyl Hydroxybenzoate joint toxicity.
The technical solution adopted for the present invention to solve the technical problems is as follows:
The method comprises the following steps:
Step (1). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (2). take out step (1) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples,
Incubation time is 72h；Set up the cell culture fluid adding 40 μ L/ holes to make negative control (i.e. to set up simultaneously
Step (1) carry cultivation after 96 orifice plates of SMMC-7721 cell discard culture fluid after add 160 μ L/
Hole RPMI-1640 culture medium, the cell culture fluid in 40 μ L/ holes, incubation time is 72h, and it is negative right to make
According to)；
Described testing sample is the reddest, Sodium Methyl Hydroxybenzoate mixed liquor, and ultimate density is according to food
Dosage in product packaging bag adds；
Step (3). take out 96 orifice plates after step (2) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Described live cell dye solution is Hoechst 34580, Mitotracker orange,
The mixed liquor of lysoTracker tri-kinds of dyestuffs of Deep Red, the ultimate density ratio of three is 1: 2: 1；
Step (4). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is the paraformaldehyde of mass percent 4%；
Step (5). the PBS adding 100 μ L/ holes in 96 orifice plates after step (4) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Described permeable membrane buffer is the PBS containing mass percent 0.1%Triton X-100；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes washes
After washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio), often
Temperature lucifuge hatches 1h；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes washes
After washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become image height
Intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection Hoechst
The nucleus of 34580 labellings, second channel selects the cellular microtubules egg of " FITC " detection FITC labelling
In vain, third channel selects the mitochondrial membrane potential of " Cy3 " detection Mitotracker orange labelling (to live
Cell), fourth lane selects the lysosome PH of " Fura-2 " detection lysoSensor Blue labelling
Value；Total cellular score is detected under ordinary light source；Selecting 20 times of object lens, every hole gathers the figure in 10 visuals field
Picture, storage information is for graphical analysis；
Step (8). employing MetaXpress analyzes total cellular score, the work that step (7) is detected by software
Cell quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out fixed
Position and quantitative analysis, and with total cellular score, living cells quantity, nucleus state, cellular tubulin,
Mitochondrial membrane potential, lysosome PH parametric form represent, according to MetaXpress analysis software
Total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, lyase
Six parameter decision food additive of body PH are the reddest, the joint toxicity of Sodium Methyl Hydroxybenzoate.
Judge that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage are used in combination cytotoxic standard:
Lysosome PH and negative control group (adding cell culture fluid group) significant difference (P ＜ 0.05), and
Arbitrary index in other five indexs or multiple index and negative control group significant difference (P ＜ 0.05),
I.e. can determine that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage have been used in combination cytotoxicity.Note: as
Really total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential five
Arbitrary index in index or multiple index and negative control group significant difference (P ＜ 0.05), and lysosome
PH and negative control group difference is not notable (P ＞ 0.05), and this detection is invalid, needs again to enter sample
Row detection；If total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrion
Transmembrane potential, six indexs of lysosome PH are not all notable (P ＞ 0.05) with negative control group difference, then table
The reddest, the Sodium Methyl Hydroxybenzoate of this dosage bright are used in combination no cytotoxicity；If lysosome PH
Single index and negative control group significant difference (P ＜ 0.05), then show the reddest, to hydroxyl of this dosage
Essence of Niobe sodium is used in combination has potential cytotoxicity, needs to be analyzed further identifying.
Beneficial effects of the present invention: the present invention is directed to that food additive is the reddest, Sodium Methyl Hydroxybenzoate
Chemical combination Structure Selection characterize six spies of associational cells toxicity of the reddest, Sodium Methyl Hydroxybenzoate
Opposite sex index, has preferable specificity；The present invention has carried out repeated checking, utilizes chessboard method to do
100 concentration combination, each combination is repeated 3 times, it was demonstrated that the method has preferable repeatability；This
Invention has carried out blend proportion optimization according to the characteristic of cell dye, so that commonsense method needs to carry out four
Experimental index that secondary operation just can complete and now only need operation once, when making amount of reagent, detection
Between etc. aspect be all greatly reduced, decrease the probability made mistakes to a certain extent；The present invention establishes quickly
Detection food additive is the reddest, the associational cells toxicity method of Sodium Methyl Hydroxybenzoate, can be food
Product additive is the reddest, the proper use of offer theoretical foundation of Sodium Methyl Hydroxybenzoate, to ensureing food
Proper use of and the food safety of additive is significant.
Detailed description of the invention
For further analysis to the present invention below in conjunction with specific embodiment.
Embodiment 1
Step (1). gather XXX board fruit-flavored beverage to supermarket, record adds the reddest, P-hydroxybenzoic acid
The dosage of methyl ester sodium, respectively 0.05g/kg, 0.16g/kg.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), the reddest, the interpolation of Sodium Methyl Hydroxybenzoate
Amount is respectively 0.05g/kg, 0.16g/kg, and incubation time is 72h；Set up addition 40 μ L/ simultaneously
The cell culture fluid in hole makees negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, interpolation 0.05g/kg is the reddest, 0.16g/kg
After Sodium Methyl Hydroxybenzoate, total cellular score, living cells quantity, nucleus state, cellular microtubules egg
In vain, mitochondrial membrane potential, lysosome PH and negative control there was no significant difference (P ＞ 0.05), show
It is no cytotoxicity that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage combines interpolation.
Embodiment 2
Step (1). gather XXX board beverage to supermarket, record that its interpolation is the reddest, P-hydroxybenzoic acid first
The dosage of ester sodium, respectively 0.05g/kg, 0.18g/kg.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), the reddest, the interpolation of Sodium Methyl Hydroxybenzoate
Amount is respectively 0.05g/kg, 0.18g/kg, and incubation time is 72h；Set up addition 40 μ L/ simultaneously
The cell culture fluid in hole makees negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, interpolation 0.05g/kg is the reddest, 0.18g/kg
After Sodium Methyl Hydroxybenzoate, total cellular score, living cells quantity, nucleus state, cellular microtubules egg
In vain, mitochondrial membrane potential, lysosome PH and negative control there was no significant difference (P ＞ 0.05), show
It is no cytotoxicity that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage combines interpolation.
Embodiment 3
Step (1). gather XXX board beverage to supermarket, record that its interpolation is the reddest, P-hydroxybenzoic acid first
The dosage of ester sodium, respectively 0.05g/kg, 0.20g/kg.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), the reddest, the interpolation of Sodium Methyl Hydroxybenzoate
Amount is respectively 0.05g/kg, 0.20g/kg, and incubation time is 72h；Set up addition 40 μ L/ simultaneously
The cell culture fluid in hole makees negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, interpolation 0.05g/kg is the reddest, 0.20g/kg
After Sodium Methyl Hydroxybenzoate, total cellular score, living cells quantity, nucleus state, cellular microtubules egg
In vain, mitochondrial membrane potential, lysosome PH and negative control there was no significant difference (P ＞ 0.05), show
It is no cytotoxicity that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage combines interpolation.
Embodiment 4
Step (1). gather XXX board confection and beverage to supermarket, it is the reddest, right to record after both being mixed
The dosage of methyl hydroxybenzoate sodium, respectively 0.05g/kg, 0.21g/kg.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), the reddest, the interpolation of Sodium Methyl Hydroxybenzoate
Amount is respectively 0.05g/kg, 0.21g/kg, and incubation time is 72h；Set up addition 40 μ L/ simultaneously
The cell culture fluid in hole makees negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, interpolation 0.05g/kg is the reddest, 0.21g/kg
After Sodium Methyl Hydroxybenzoate, total cellular score, living cells quantity, nucleus state, cellular microtubules egg
In vain, mitochondrial membrane potential, lysosome PH and negative control there was no significant difference (P ＞ 0.05), show
It is no cytotoxicity that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage combines interpolation.
Embodiment 5
Step (1). the beverage that laboratory makes, record the reddest last, Sodium Methyl Hydroxybenzoate
Dosage, respectively 1.2g/L, 0.8g/L.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), end the reddest, Sodium Methyl Hydroxybenzoate is dense
Degree is respectively 1.2g/L, 0.8g/L, and incubation time is 72h；Set up simultaneously and add 40 μ L/ holes
Cell culture fluid makees negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, adds that 1.2g/L is the reddest, 0.8g/L is to hydroxyl
After yl benzoic acid methyl ester sodium, total cellular score, cellular tubulin, nucleus state, mitochondrial membrane potential,
Lysosome PH and negative control there was no significant difference (P ＞ 0.05), total viable cell and negative control group
Significant difference (P ＜ 0.05).Owing to lysosome PH index and negative control there was no significant difference (P ＞
0.05), according to criterion, this detection is invalid, needs again to detect sample；Again examine
Survey result shows, adds that 1.2g/L is the reddest, after 0.8g/L Sodium Methyl Hydroxybenzoate, and cell is total
Number, living cells quantity, nucleus state, mitochondrial membrane potential there was no significant difference (P with negative control
＞ 0.05), cellular tubulin, lysosome PH and negative control significant difference (P ＜ 0.05), show
It is cytotoxic that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage combines interpolation.
Embodiment 6
Step (1). the beverage that laboratory makes, record the reddest last, Sodium Methyl Hydroxybenzoate
Dosage, respectively 0.62g/kg, 0.67g/kg.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), the reddest, the interpolation of Sodium Methyl Hydroxybenzoate
Amount is respectively 0.62g/kg, 0.67g/kg, and incubation time is 72h；Set up addition 40 μ L/ simultaneously
The cell culture fluid in hole makees negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, interpolation 0.62g/kg is the reddest, 0.67g/kg
After Sodium Methyl Hydroxybenzoate, total cellular score, living cells quantity, nucleus state, cellular microtubules shape
State, mitochondrial membrane potential and negative control there was no significant difference (P ＞ 0.05), and lysosome PH is with cloudy
Property contrast difference notable (P ＜ 0.05), show the reddest, the Sodium Methyl Hydroxybenzoate associating of this dosage
Interpolation is to have potential Cytotoxic, needs further analysis verification.
Embodiment 7
Step (1). the beverage that laboratory makes, record the reddest last, Sodium Methyl Hydroxybenzoate
Dosage, respectively 1.1g/kg, 1.2g/kg.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), the reddest, the interpolation of Sodium Methyl Hydroxybenzoate
Amount is respectively 1.1g/kg, 1.2g/kg, and incubation time is 72h；Set up addition 40 μ L/ holes simultaneously
Cell culture fluid make negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, add 1.1g/kg the reddest, 1.2g/kg pair
After methyl hydroxybenzoate sodium, total cellular score, living cells quantity, nucleus state, mitochondrial membrane potential
There was no significant difference with negative control (P ＞ 0.05), and cellular microtubules state, lysosome PH are with negative
Contrast difference is notable (P ＜ 0.05), shows that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage are combined and adds
It is cytotoxic for adding.
Embodiment 8
Step (1). the beverage that laboratory makes, record the reddest last, Sodium Methyl Hydroxybenzoate
Dosage, respectively 2.1g/kg, 1.2g/kg.
Step (2). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell (people's hepatocarcinoma
Cell) with 5 × 104Individual/hole/100 μ L kind plate, 37 DEG C, 5%CO2Cultivate 12h；The cell training used
Nutrient solution is RPMI-1640 culture medium；
Step (3). take out step (2) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard
Culture fluid, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples
(the reddest, Sodium Methyl Hydroxybenzoate mixed liquor), the reddest, the interpolation of Sodium Methyl Hydroxybenzoate
Amount is respectively 2.1g/kg, 1.2g/kg, and incubation time is 72h；Set up addition 40 μ L/ holes simultaneously
Cell culture fluid make negative control；
Step (4). take out 96 orifice plates after step (3) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Step (5). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；Wherein fixative is mass percent 4% paraformaldehyde；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes enters
After row washing 2 times, add the cellular tubulin antibody of the FITC labelling of 1: 1000 (volume ratio),
Room temperature lucifuge hatches 1h.
Step (8). the PBS adding 100 μ L/ holes in 96 orifice plates after step (7) processes enters
After row washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become
Image height intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection
The nucleus of Hoechst 34580 labelling, second channel selects the thin of " FITC " detection FITC labelling
Born of the same parents' tubulin, third channel selects the mitochondrion of " Cy3 " detection Mitotracker orange labelling
Transmembrane potential (living cells), fourth lane selects the molten of " Fura-2 " detection lysoSensor Blue labelling
Enzyme body pH value；Total cellular score is detected under ordinary light source.Selecting 20 times of object lens, every hole gathers 10
The image in the visual field, storage information is for graphical analysis；
Step (9). use MetaXpress to analyze software to total cellular score, living cells quantity, cell
Nuclear state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out positioning and quantitative analysis,
And with total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential,
Lysosome PH parametric form represents, total cellular score, work according to MetaXpress analysis software are thin
Born of the same parents' quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, six parameters of lysosome PH
Judge the joint toxicity of food additive.Result shows, add 2.1g/kg the reddest, 1.2g/kg pair
After methyl hydroxybenzoate sodium, total cellular score and negative control there was no significant difference (P ＞ 0.05), and live
Cell quantity, nucleus state, mitochondrial membrane potential, cellular microtubules state, lysosome PH are with negative
Contrast difference is notable (P ＜ 0.05), shows that the reddest, the Sodium Methyl Hydroxybenzoate of this dosage are combined and adds
It is cytotoxic for adding.
Claims (4)
1. quick detection food additive (the reddest, a Sodium Methyl Hydroxybenzoate) joint toxicity
Method, it is characterised in that the method comprises the following steps:
Step (1). on 96 orifice plates overlaying cell adhesion agent, SMMC-7721 cell is with 5 × 104
Individual/hole/100 μ L kind plate, 37 DEG C, 5% CO2Cultivate 12h；
Step (2). take out step (1) and carry 96 orifice plates of SMMC-7721 cell after cultivation, discard training
Nutrient solution, is subsequently adding 160 μ L/ hole RPMI-1640 culture medium, 40 μ L/ hole 5 × testing samples,
Incubation time is 72h；Set up the cell culture fluid adding 40 μ L/ holes to make negative control simultaneously；
Described testing sample is the reddest, Sodium Methyl Hydroxybenzoate mixed liquor, and ultimate density is according to food
Dosage in product packaging bag adds；
Step (3). take out 96 orifice plates after step (2) is cultivated, add the live cell dye in 50 μ L/ holes
Solution, hatches 30min for 37 DEG C；
Described live cell dye solution is Hoechst 34580, Mitotracker orange,
The mixed liquor of lysoTracker tri-kinds of dyestuffs of Deep Red, the ultimate density ratio of three is 1: 2: 1；
Step (4). discard culture fluid, add the fixative of the 100 pre-temperature in μ L/ hole to 37 DEG C, room temperature lucifuge
Place 20min；
Step (5). the PBS adding 100 μ L/ holes in 96 orifice plates after step (4) processes is carried out
Washing 1 time, add the permeable membrane buffer in 100 μ L/ holes, room temperature lucifuge hatches 10min；
Step (6). the PBS adding 100 μ L/ holes in 96 orifice plates after step (5) processes washes
After washing 2 times, adding the cellular tubulin antibody of 1000 times of volume FITC labellings, room temperature lucifuge is incubated
Educate 1h；
Step (7). the PBS adding 100 μ L/ holes in 96 orifice plates after step (6) processes washes
After washing 2 times, add the PBS in 200 μ L/ holes, then use ImageXpress Micro to become image height
Intension image analysis system detects, and testing conditions is as follows: first passage selects " DAPI " detection Hoechst
The nucleus of 34580 labellings, second channel selects the cellular microtubules egg of " FITC " detection FITC labelling
In vain, third channel selects the mitochondrial membrane potential of " Cy3 " detection Mitotracker orange labelling (to live
Cell), fourth lane selects the lysosome PH of " Fura-2 " detection lysoSensor Blue labelling
Value；Total cellular score is detected under ordinary light source；Selecting 20 times of object lens, every hole gathers the figure in 10 visuals field
Picture, storage information is for graphical analysis；
Step (8). employing MetaXpress analyzes total cellular score, the work that step (7) is detected by software
Cell quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, lysosome PH carry out fixed
Position and quantitative analysis, and with total cellular score, living cells quantity, nucleus state, cellular tubulin,
Mitochondrial membrane potential, lysosome PH parametric form represent, according to MetaXpress analysis software
Total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane potential, lyase
Six parameter decision food additive of body PH are the reddest, the joint toxicity of Sodium Methyl Hydroxybenzoate.
2. one as claimed in claim 1 quickly detects food additive (the reddest, para hydroxybenzene first
Acid methyl ester sodium) joint toxicity method, it is characterised in that step (4) wherein fixative is mass percent 4%
Paraformaldehyde.
3. one as claimed in claim 1 quickly detects food additive (the reddest, para hydroxybenzene first
Acid methyl ester sodium) joint toxicity method, it is characterised in that the permeable membrane buffer described in step (5) is containing quality
The PBS of percentage ratio 0.1%Triton X-100.
4. one as claimed in claim 1 quickly detects food additive (the reddest, para hydroxybenzene first
Acid methyl ester sodium) joint toxicity method, it is characterised in that step (8) judges the reddest under certain dosage, to hydroxyl
It is as follows that yl benzoic acid methyl ester sodium is used in combination cytotoxic standard:
If 1. lysosome PH and negative control group difference is not notable (P ＞ 0.05), but total cellular score,
Arbitrary in living cells quantity, nucleus state, cellular tubulin, five indexs of mitochondrial membrane potential
Index or multiple index and negative control group significant difference (P ＜ 0.05), then this detection is invalid, needs
Again sample is detected；
If 2. lysosome PH and negative control group significant difference (P ＜ 0.05), and total cellular score, work are carefully
Arbitrary index in born of the same parents' quantity, nucleus state, cellular tubulin, five indexs of mitochondrial membrane potential
Or multiple index and negative control group significant difference (P ＜ 0.05), then judge the reddest under this dosage, to hydroxyl
Yl benzoic acid methyl ester sodium has been used in combination cytotoxicity；
If 3. total cellular score, living cells quantity, nucleus state, cellular tubulin, mitochondrial membrane electricity
Position, six indexs of lysosome PH are not all notable (P ＞ 0.05) with negative control group difference, then show this
The reddest, the Sodium Methyl Hydroxybenzoate of dosage are used in combination no cytotoxicity；
If 4. the single index of lysosome PH and negative control group significant difference (P ＜ 0.05), then show this
The reddest, the Sodium Methyl Hydroxybenzoate of dosage are used in combination has potential cytotoxicity, needs into one
Step carries out Analysis and Identification.
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| US20050002552A1 (en) * | 2003-04-30 | 2005-01-06 | Pfizer Inc | Automated in vitro cellular imaging assays for micronuclei and other target objects |
| CN101784895A (en) * | 2007-06-26 | 2010-07-21 | 协乐民公司 | Method for predicting biological systems responses in hepatocytes |
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|---|---|---|---|---|
| US20050002552A1 (en) * | 2003-04-30 | 2005-01-06 | Pfizer Inc | Automated in vitro cellular imaging assays for micronuclei and other target objects |
| CN101784895A (en) * | 2007-06-26 | 2010-07-21 | 协乐民公司 | Method for predicting biological systems responses in hepatocytes |
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