CN101603068A - The application of Hematorylin in measuring cell-proliferation activity and medicine pair cell toxic effect - Google Patents
The application of Hematorylin in measuring cell-proliferation activity and medicine pair cell toxic effect Download PDFInfo
- Publication number
- CN101603068A CN101603068A CNA2009100162503A CN200910016250A CN101603068A CN 101603068 A CN101603068 A CN 101603068A CN A2009100162503 A CNA2009100162503 A CN A2009100162503A CN 200910016250 A CN200910016250 A CN 200910016250A CN 101603068 A CN101603068 A CN 101603068A
- Authority
- CN
- China
- Prior art keywords
- cell
- hole
- hematorylin
- minutes
- value
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses Hematorylin measuring attached cell proliferation activity and medicine, utilize the Hematorylin pair cell to dye, measure the absorbance under the specific wavelength then, thereby draw corresponding data and conclusion the application in the attached cell toxic action.The contriver adopt the haematoxylin dyeing method to SGC7901, M21 and attached cells such as Huvec not simultaneously not mutually the repeatedly measurement result of (12h, 24h, 48h, 72h) cell-proliferation activity all show, this method is the specificity height not only, and CV＜3% has repeatable preferably.That the present invention has is highly sensitive, detected result stable, applied widely, low cost and other advantages.
Description
Technical field
The present invention relates to Hematorylin and measuring attached cell proliferation activity and medicine the application in the attached cell toxic action.
Background technology
Deep day by day along with antitumor drug exploitation, the tumor-inhibiting action of more and more many Chinese medicines is paid close attention to by common people, and this class medicine or its extract are carried out the important step that in-vitro screening also is a drug discovery process.Employing is stable, reliable detection method observation medicine is essential to different clone in-vitro multiplications inhibition activity.
Observation of cell proliferation activity and cytotoxic effect, conventional four class methods that adopt: 1, isotope method mainly contains
3H-TdR mix method,
125I-UdR reaches
51The Cr method for releasing is though there is environmental pollution in this class methods sensitivity, special and needs many drawbacks such as plant and instrument of complex and expensive.2, the flow cytometry analysis method has sensitivity, advantage such as special equally, but also must expensive complicated plant and instrument, is difficult for applying in basic unit.3, Giemsa staining, Viola crystallina, platform are expected stainings such as orchid, though easy, economical, there are unstable result and serious shortcomings such as non-specific adsorption.4, the enzyme substrates method mainly contains traditional MTT colorimetry etc., be characterized in coming showed cell quantity and survival condition with certain enzyme of viable cell and the reaction between the corresponding substrate, advantage is fast responsive and better repeated, and to the human zero damage, by perfect, be widely used in research fields such as cell proliferation and cytotoxic effect for many years to updating of this method always.CCK-8 is a kind of upgrading substitute products of MTT, and its action principle is similar to MTT, compares with MTT that to have susceptibility higher, and advantage such as operation is easier, is suitable for very much the cytotoxicity experiment of cell proliferation and general chemicals.This research when adopting MTT colorimetry and CCK-8 colorimetry to observe the Chinese medical extract Wild Buckwheat Rhizome, find the toxic action of tumour cell MTT and CCK-8 all with Rhizoma Fagopyri Dibotryis extract generation nonspecific reaction in various degree, cause that fluctuation range is bigger as a result, dose-dependence is not obvious.Based on above-mentioned described, seek special, stable, easy, economic external drug screening method and become the task of top priority.
Hematorylin is a kind of natural dyestuff, becomes haematein by oxidation, is generally used for the nuclear dyeing of biological tissue section.This research is used to detect attached cell propagation and medicine to the attached cell toxic action with Hematorylin, the viable cell absorption before Hematorylin only can be fixed, viable cell quantity is many more, state is good more, it is dark more then to dye, the OD value of surveying high more; Otherwise viable cell quantity is few more, and state is poor more, and then dyeing is shallow more, and the OD value is low more, thereby indirectly the quantity of viable cell is carried out quantitatively.At present still no-trump its to be used to measure attached cell be research and the report of the active and medicine of in-vitro multiplication to the attached cell toxic action.
Summary of the invention
At above-mentioned prior art, the invention provides haematoxylin a kind of new purposes, promptly Hematorylin is being measured attached cell proliferation activity and medicine to the application in the attached cell toxic action.The present invention has also set up a cover and has measured attached cell proliferation activity and the medicine detection method to the attached cell toxic action.Special, stable, easy, economic dispatch advantage that detection method of the present invention has.
The present invention is achieved by the following technical solutions:
A kind of method of utilizing Hematorylin to measure the attached cell proliferation activity, step is as follows: get and dilute good cell suspension to be measured, place each respective aperture of Tissue Culture Plate, every hole 200 μ l are 5% CO in 37 ℃, volume fraction
2Under the saturated humidity condition, hatched 44 hours, get rid of and abandon supernatant, add 4% paraformaldehyde solution immediately, every hole 100 μ l collect stationary liquid (can recycle) after fixing 20 minutes, and PBS washes 3 times, and supernatant is abandoned in each 200 μ l/ holes, and room temperature is dried; Get haematoxylin dyeing liquid, every hole adds 100 μ l, collects staining fluid (can recycle) after 20 minutes, distillation washing 3～5 times, and to remove unconjugated dyestuff, room temperature is dried; The painted situation of observation of cell under inverted microscope, add 1% hydrochloride alcohol again, 100 μ l/ holes, this moment, each hole liquid presented pink in various degree, measure the absorbance and the record at 540nm place in 20 minutes with microplate reader, get and respectively organize OD value mean and draw the cell line proliferation curve.
A kind of Hematorylin that utilizes is measured the method for medicine to the tumor cell line toxic action, step is as follows: get enchylema to be measured, place the orifice plate of Tissue Culture Plate, every hole 200 μ l, setting no drug cell contrast and the substratum microplate reader hole of returning to zero simultaneously, is 5% CO in 37 ℃, volume fraction
2Under the saturated humidity condition, hatched 44 hours; Take out Tissue Culture Plate and get rid of and abandons supernatant, add 4% paraformaldehyde solution immediately, stationary liquid is collected after fixing 20 minutes in 150 μ l/ holes; PBS washes 3 times, and supernatant is abandoned in each 200 μ l/ holes, and room temperature is dried; Get haematoxylin dyeing liquid, every hole adds 100 μ l, collects staining fluid after 20 minutes; Distillation is washed 3～5 times to remove unconjugated dyestuff, and room temperature is dried; The painted situation of observation of cell under inverted microscope, add 1% hydrochloride alcohol again, 100 μ l/ holes, this moment, each hole liquid presented pink in various degree, measure the absorbance and the record at 540nm place in 20 minutes with microplate reader, be calculated as follows the tumour inhibiting rate of medicine:
Wherein, the mean of each hole OD value of OD value (test holes X) expression experimental group; The mean of no each the hole OD value of medicine cell control group of OD value (cell control well X) expression.
Described 4% paraformaldehyde solution is that (levy in Hubei Province according to document " tissue culture and cytologic technology ", the P146 of Beijing Publishing House) the description preparation in, method is as follows: Paraformaldehyde 96 40g is dissolved among the 0.1mol PBS 1000ml, be heated to about 60 ℃, constantly be stirred to transparently, also can add a little 1mol NaOH and make solution transparent.
Described haematoxylin dyeing liquid is by document " comparison of several prescriptions of Hematorylin commonly used and coloration result " (Li Wen, Zhang Saixia, anatomy magazine, 2000; 23 (4): the description preparation 393), method is as follows: 1.5g Hematorylin 5ml anhydrous alcohol solution, the dissolved in distilled water potassium alum (is heated to 90 ℃, do not boil), dissolved Hematorylin and sodium iodate are added in the potassium alum aqueous solution simultaneously, and flowing water is cooled to room temperature, again glycerol adding, shake and can use after 1 hour, dye liquor can be guaranteed 6 months.
Described 1% hydrochloride alcohol solution is meant that hydrochloric acid and alcohol volume fraction are respectively 1% and 70% solution.
Described PBS is meant the phosphate buffered saline buffer of 0.01mol/L, PH=7.2.
The paraformaldehyde solution of described adding 4%, PBS and haematoxylin dyeing liquid are to adopt the hyperchannel micropipet, not only can simplify loaded down with trivial details experimental arrangement, and can significantly reduce the error between each hole.
The present inventor adopt the haematoxylin dyeing method to SGC7901, M21 and attached cells such as Huvec not simultaneously not mutually the repeatedly measurement result of (12h, 24h, 48h, 72h) cell-proliferation activity all show, this method is the specificity height not only, and CV＜3% has repeatable preferably.
The contriver is adopting MTT colorimetry and CCK-8 colorimetry to observe the multiplication effect of grape pip procyanidin (GSPE) pair cell, Rhizoma Fagopyri Dibotryis extract is done the time spent to cytotoxicity and is found, MTT and CCK-8 all can produce in various degree nonspecific reaction with this type of Chinese medical extract, detected result (OD value) with do not mix MTT and CCK-8 before the microscopy observations differ greatly, dose-dependence is not obvious, causes that fluctuation range is bigger as a result.And the haematoxylin dyeing method is applied to the toxicity test of Rhizoma Fagopyri Dibotryis extract to tumour cell (M21 and SGC7901 cell), repeatedly experimental result shows that M21 and SGC7901 cell have dose-dependence preferably to Rhizoma Fagopyri Dibotryis extract, and consistent with microscopy observations height, prompting this method can reflect the cytotoxicity of this type of material to different cells more objectively.
Detection method of the present invention is after fixing and dyeing finish, and stationary liquid and haematoxylin dyeing liquid are recyclable and repeatedly use repeatedly.Compare with the CCK-8 reagent of costliness, the economical and practical of haematoxylin dyeing method then seems rather outstanding.Detection method of the present invention has advantages such as effective, that cost is low, detected result is stable.
Description of drawings
Fig. 1 is haematoxylin dyeing method and the mtt assay comparison synoptic diagram to the mensuration of Huvec proliferation activity;
Fig. 2 is haematoxylin dyeing method and the CCK-8 method comparison synoptic diagram to the mensuration of Huvec proliferation activity;
Fig. 3 is haematoxylin dyeing method and the mtt assay comparison synoptic diagram to the mensuration of M21 cell-proliferation activity;
Fig. 4 is the toxic action synoptic diagram of VCR to the M21 cell;
Fig. 5 is the toxic action synoptic diagram of VCR to the SGC7901 cell;
Fig. 6 is the toxic action synoptic diagram of 5-Fu to the M21 cell;
Fig. 7 is the toxic action synoptic diagram of 5-Fu to the SGC7901 cell;
Fig. 8 is the toxic action synoptic diagram of cis-platinum to the VX2 cell;
Fig. 9 is that haematoxylin dyeing method and CCK-8 method are measured the toxic action synoptic diagram of Rhizoma Fagopyri Dibotryis extract to the SGC7901 cell;
Figure 10 measures the toxic action synoptic diagram of Rhizoma Fagopyri Dibotryis extract to the SGC7901 cell for the haematoxylin dyeing method;
Figure 11 is that haematoxylin dyeing method and CCK-8 method are measured the toxic action synoptic diagram of Rhizoma Fagopyri Dibotryis extract to the M21 cell;
Figure 12 measures the toxic action synoptic diagram of Rhizoma Fagopyri Dibotryis extract to M21 for the CCK-8 method;
Figure 13 is the toxic action synoptic diagram (100mg/L of Rhizoma Fagopyri Dibotryis extract to the M21 cell; Amplify 250 times);
Figure 14 is the toxic action synoptic diagram (0mg/L of Rhizoma Fagopyri Dibotryis extract to the M21 cell; Amplify 250 times).
Embodiment
The present invention is further illustrated below in conjunction with embodiment:
Embodiment 1:
(1) the haematoxylin dyeing method detects the attached cell proliferation activity, and is as follows:
Get 1640 perfect mediums and add in the 96 porocyte culture plates, 100 μ l/ holes.0.25% pancreatin digests Huvec and M21 cell respectively, abandons supernatant after centrifugal, transfers corresponding cell concn with perfect medium.Respectively getting 100 μ l cell suspensions then adds respectively in above-mentioned 96 each respective aperture of porocyte culture plate, 8 extent of dilution are established in multiple proportions 2 dilutions successively behind the mixing, each extent of dilution 3 multiple hole, each hole replenishes perfect medium to 200 μ l then, and establishes 1 hole, 1640 substratum and be used for the microplate reader zeroing.It is 5% CO that 96 porocyte culture plates are put 37 ℃, volume fraction
2Under the saturated humidity condition, hatched 44 hours.Take out 96 porocyte culture plates and get rid of and abandon supernatant, add 4% paraformaldehyde solution immediately with 8 passage micropipets, stationary liquid (can use repeatedly) is collected after fixing 20 minutes in 150 μ l/ holes.PBS washes 3 times, abandons supernatant, and room temperature is dried.Get haematoxylin dyeing liquid 100 μ l with 8 passage micropipets and add each hole, collect staining fluid (can recycle) after 20 minutes.Distillation is washed 3-5 time to remove unconjugated dyestuff, and room temperature is dried.The painted situation of observation of cell under inverted microscope, the hydrochloride alcohol of adding 1%, 100 μ l/ holes, this moment, each hole liquid presented pink in various degree, measured the absorbance and the record at 540nm place in 20 minutes with microplate reader.Calculate and respectively organize OD value mean and draw the cell line proliferation curve.
(2) the MTT colorimetry is observed the proliferation activity of Huvec and M21 clone, presses document Tian Zhigang, the application China tumor biotherapy magazine 1994 of Zhang Jianhua mtt assay in detecting cell proliferation and cellulotoxic effect; 1 (1): 74 methods are carried out, and step slightly.
(3) the CCK-8 colorimetry is observed the proliferation activity of Huvec, and is as follows:
At first get 100 μ l perfect mediums and add each hole.0.25% trysinization is transferred corresponding cell concn with perfect medium after collecting Huvec.Behind the cell suspension mixing, to get 100 μ l respectively and add in 96 each respective aperture of porocyte culture plate, 8 extent of dilution are established in multiple proportions 2 dilution behind the mixing, each concentration 3 multiple hole, and establish the 1640 substratum contrast of 1 hole and be used for the microplate reader zeroing.96 porocyte culture plates are put 37 ℃, 5% CO
2Cultivated 44 hours under the saturated humidity condition.Get CCK-8 solution 10 μ l and add each hole, after continuing to hatch 1h, microplate reader is measured the 450nm OD of place value and record.Calculate the mean of each concentration OD value and draw the cell proliferation curve.
Result: measure proliferation activity after Huvec is hatched 44 hours with above-mentioned three kinds of methods, the cell proliferation curve of drawing more as shown in Figure 1 and Figure 2, though three kinds of measuring methods need be selected three different wave lengths (570nm, 540nm and 450nm), and the OD value of surveying differs bigger, especially the highest OD value of haematoxylin dyeing method only is 0.27, CCK-8 colorimetry OD value is then near 3.0, not only linear relationship is good but three kinds of methods are surveyed the OD value, and has similar response curve (seeing Fig. 1,2).
Haematoxylin dyeing method and mtt assay to the M21 cell-proliferation activity more as shown in Figure 3.Haematoxylin dyeing method and MTT colorimetric method for determining result demonstration, linear after the M21 cell is hatched 44 hours in certain density range inner cell quantity and OD value, and the M21 cell concn is 1 * 10
9During/L, big or incubation time is long owing to cell density causes that the consumption of cell desired nutritional is fast, thereby causes part necrocytosis or poor growth, then OD value (Fig. 3) on a declining curve.
Embodiment 2:
(1) haematoxylin dyeing method detection of drugs is to the toxic action of tumor cell line
Get M21, SGC7901 and VX2 cell respectively, abandon supernatant after centrifugal, transfer corresponding cell concn, add in the 96 porocyte culture plates 100 μ l/ holes successively with perfect medium with 0.25% trysinization.96 orifice plates are put 37 ℃, 5% CO
2Overnight incubation makes it adherent under the saturated humidity condition.VCR, 5-Fu, CDDP and Rhizoma Fagopyri Dibotryis extract are diluted 5 or 6 different concentration successively, add in each respective aperture of 96 orifice plates 100 μ l/ holes, 3 multiple hole/concentration respectively, other establishes the 6-9 hole does not have drug cell contrast and 1 hole, 1640 substratum microplate reader zeroing hole, and every hole total amount of liquid is 200 μ l.96 porocyte culture plates are put 37 ℃, 5% CO
2Hatched under the saturated humidity condition 44 hours.Take out 96 porocyte culture plates and get rid of and abandon supernatant, add 4% paraformaldehyde solution immediately, stationary liquid is collected after fixing 20 minutes in 150 μ l/ holes; PBS washes 3 times, abandons supernatant, and room temperature is dried; Get haematoxylin dyeing liquid, every hole adds 100 μ l, collects staining fluid after 20 minutes; Distillation is washed 3～5 times to remove unconjugated dyestuff, and room temperature is dried; The painted situation of observation of cell under inverted microscope, add 1% hydrochloride alcohol again, 100 μ l/ holes, this moment, each hole liquid presented pink in various degree, measure the absorbance and the record at 540nm place in 20 minutes with microplate reader, the tumour inhibiting rate calculation formula of different pharmaceutical is as follows:
(2) MTT colorimetry detection of drugs is to the toxic action of tumor cell line: press document Tian Zhigang, the Zhang Jianhua mtt assay is detecting cell proliferation and the Chinese tumor biotherapy magazine 1994 of the application in the cellulotoxic effect; 1 (1): the method in 74 is carried out, and step slightly.
The result: haematoxylin dyeing method and MTT colorimetric method for determining VCR, 5-Fu are to the toxic action of M21 and SGC7901 cell, and the result shows that two kinds of tumor cell lines all have dose-response relationship to medicine, and experimental repeatability is (CV＜8%) better.What is interesting is that haematoxylin dyeing tumour inhibiting rate that method is surveyed all is higher than MTT tumour inhibiting rate that colorimetry is surveyed (Fig. 4,5,6,7), this research thinks that haematoxylin dyeing method measured result is near the microscopically observations.Because the haematoxylin dyeing method only can make the viable cell before fixing painted, and dead cell and fragment can not absorbing dyes before fixing, show that this method has stronger specificity and stability.Fig. 9 shows that cis-platinum has good tumor-inhibiting action to the VX2 cell, and two kinds of method measured results present tangible dose-response relationship.
Embodiment 3:CCK-8 colorimetry detects the toxic action of Rhizoma Fagopyri Dibotryis extract to tumour cell:
Get M21, SGC7901 cell respectively, with 0.25% trysinization centrifugal after, perfect medium is transferred corresponding cell concn, adds in the 96 porocyte culture plates 100 μ l/ holes successively.96 orifice plates are put 37 ℃, 5% CO
2Overnight incubation makes it adherent under the saturated humidity condition.With 6 different concns of Rhizoma Fagopyri Dibotryis extract dilution, add successively in 96 each respective aperture of porocyte culture plate, 100 μ l/ holes, 3 multiple hole/concentration, other establishes no drug cell and contrasts 6 holes and 1 hole, 1640 substratum microplate reader zeroing hole, and every hole total amount of liquid is 200 μ l.96 porocyte culture plates are put 37 ℃, 5%CO
2Hatched in the incubator 44 hours.Take out 96 porocyte culture plates, inhale gently with micropipet and abandon 100 μ l supernatants, add CCK-8 solution 10 μ l/ holes then.37 ℃ were continued to hatch 1 hour, and microplate reader is measured the 450nm OD of place value and record result.The calculation formula of tumour inhibiting rate is with embodiment 2.
Result: adopt MTT colorimetry, CCK-8 colorimetry and haematoxylin dyeing method to measure the toxic action of Rhizoma Fagopyri Dibotryis extract to SGC7901 and M21 cell.It is a straight line substantially that the MTT colorimetry is surveyed the OD value, no dose-dependence (result is not listed as).Fig. 9 shows that the CCK-8 colorimetry shows that Rhizoma Fagopyri Dibotryis extract has stronger toxic action at 400mg/L and 200mg/L to the SGC7901 cell, the following concentration OD value of 100mg/L all is higher than no drug cell control wells, but the cell that mirror is observed down in each hole of discovery 100mg/L has about 50% dead fragmentations.Cell in 50mg/L and each experimental port of 25mg/L all has death in various degree simultaneously, and it is good not have each porocyte survival condition of medicine control group, acellular substantially death, but the OD value is on a declining curve.The haematoxylin dyeing method is surveyed the OD value, and not only linear relationship is good, and simultaneously mirror is observed each porocyte survival condition of different pharmaceutical concentration conform to survey OD value (Figure 10) down.It is similar to the SGC7901 cell to the toxic action result of M21 cell to adopt two kinds of methods to detect Rhizoma Fagopyri Dibotryis extract.Though the CCK-8 colorimetry is surveyed OD value linear relationship still can (Figure 11,12), but when Rhizoma Fagopyri Dibotryis extract is 100mg/L, though the OD value is more lower slightly than no medicine control wells, it is relatively poor that mirror is observed each hole M21 cell state down, necrocytosis obviously (Figure 13) is compared differ greatly (Figure 14) with no drug cell control wells, points out CCK-8 colorimetry and MTT colorimetry can not reflect the survival condition of cell behind the drug effect truly.Haematoxylin dyeing Rhizoma Fagopyri Dibotryis extract that method is surveyed is good to the toxic action linear relationship of M21 cell, proves that this method is stable, special, has good repeatability (CV＜8%).
The used material of above embodiment, main agents and key instrument source are as follows:
A, clone and laboratory animal:
M21 (human melanoma cell) and Huvec (Human umbilical vein endothelial cells) all can buy from American Type Culture Collecti; SGC7901 (people's adenocarcinoma of stomach cell) can buy from typical case's culture collection council of Chinese Academy of Sciences cell bank.The VX2 cell is the rabbit squamous cell carcinoma, and this laboratory is separated into individual cells with VX2 tumor tissue, and in in-vitro cultivation surplus 300 day, generation surplus the external biography 100, the cryopreservation resuscitation artifact is learned characteristic and is not seen change, and having built is that Wuhan University can provide interior generation tumor tissue.
B, main agents:
Hematorylin (Shanghai import packing) is pressed document Li Wen, the comparative anatomy magazine of Zhang Saixia several prescriptions of Hematorylin commonly used and coloration result, 2000; 23 (4): 393 preparation Carazzi haematoxylin dyeing liquid;
1% hydrochloride alcohol solution, hydrochloric acid and alcohol volume fraction are respectively 1% and 70%;
4% Paraformaldehyde 96 is prepared by document " tissue culture and cytologic technology " (levy in Hubei Province, the P146 of Beijing Publishing House) method;
MTT (tetramethyl-azo azoles salt, Fluka product) is 5g/L with physiological saline solution, filtration sterilization;
Volume fraction is 10% SDS (sodium lauryl sulphate, the Sigma product) aqueous solution;
CCK-8, Japanese colleague's chemistry institute product;
RPMI1640 substratum (GiBCO product), the by specification preparation, including volume fraction is 10% calf serum (homemade) or foetal calf serum (GiBCO product), is perfect medium (CM);
Pancreatin Hanks solution is that 0.25% pancreatin is dissolved in the Hanks solution with volume fraction;
Vincristine(VCR) (Zhejiang Haizheng Pharmaceutical Co company limited), 5 FU 5 fluorouracil (Shanghai Xudong Hipu Medicine Co., Ltd), cis-platinum (Shandong Province Qilu Pharmaceutical Factory), all with the extremely corresponding concentration of physiological saline solution ,-20 ℃ are frozen standby;
Rhizoma Fagopyri Dibotryis extract (Qujing, Yunnan Gree health biotechnology Development Co., Ltd), behind 20% the dissolve with ethanol solution-20 ℃ frozen standby.
C, key instrument:
BioRad550 microplate reader (U.S. BioRad company product), CO
2Incubator (Japanese ESPEC), 8 passage micropipets (French Gilson Inc product), 96 porocyte culture plates (Costar company), 100 order stainless (steel) wires etc., all the other are slightly.
At last, it may be noted that: the haematoxylin dyeing method still is in the starting stage in the application in this field, the maximum OD value of measurement result is generally below 1.0, there is the narrower problem of linearity range, how further to improve viable cell to the adsorptive power of dyestuff or seek more suitably that destainer strengthens colored intensity, further improving its sensitivity is the key point that need solve from now on.In addition, when application haematoxylin dyeing method was carried out above-mentioned experiment, strict control initiator cell quantity, cell incubation time, cell fixation, dyeing and bleaching time etc. also were the primary conditions that obtains satisfied experimental result.
Claims (3)
1. the application of Hematorylin in measuring cell-proliferation activity and medicine pair cell toxic effect.
2. method of utilizing Hematorylin to measure the attached cell proliferation activity is characterized in that step is as follows: get and dilute good cell suspension to be measured, place each corresponding orifice plate of Tissue Culture Plate, every hole 200 μ l are 5% CO in 37 ℃, volume fraction
2Under the saturated humidity condition, hatched 44 hours, get rid of and abandon supernatant, add 4% paraformaldehyde solution immediately, every hole 100 μ l collect stationary liquid after fixing 20 minutes, and PBS washes 3 times, abandons supernatant, and room temperature is dried; Get haematoxylin dyeing liquid, every hole adds 100 μ l, collects staining fluid after 20 minutes, distillation washing 3～5 times, and to remove unconjugated dyestuff, room temperature is dried; The painted situation of observation of cell under inverted microscope, add 1% hydrochloride alcohol again, 100 μ l/ holes, this moment, each hole liquid presented pink in various degree, measure the absorbance and the record at 540nm place in 20 minutes with microplate reader, get and respectively organize OD value mean and draw the cell line proliferation curve.
3. one kind is utilized Hematorylin to measure the method for medicine to the tumor cell line toxic action, it is characterized in that, step is as follows: get enchylema to be measured, place the orifice plate of Tissue Culture Plate, every hole 200 μ l, setting no drug cell contrast and the substratum microplate reader hole of returning to zero simultaneously, is 5% CO in 37 ℃, volume fraction
2Under the saturated humidity condition, hatched 44 hours; Take out Tissue Culture Plate and get rid of and abandons supernatant, add 4% paraformaldehyde solution immediately, stationary liquid is collected after fixing 20 minutes in 150 μ l/ holes; PBS washes 3 times, abandons supernatant, and room temperature is dried; Get haematoxylin dyeing liquid, every hole adds 100 μ l, collects staining fluid after 20 minutes; Distillation is washed 3～5 times to remove unconjugated dyestuff, and room temperature is dried; The painted situation of observation of cell under inverted microscope, add 1% hydrochloride alcohol again, 100 μ l/ holes, this moment, each hole liquid presented pink in various degree, measure the absorbance and the record at 540nm place in 20 minutes with microplate reader, be calculated as follows the tumour inhibiting rate of medicine:
Wherein, the mean of each hole OD value of OD value (test holes X) expression experimental group; The mean of no each the hole OD value of medicine cell control group of OD value (cell control well X) expression.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100162503A CN101603068B (en) | 2009-06-23 | 2009-06-23 | Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100162503A CN101603068B (en) | 2009-06-23 | 2009-06-23 | Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101603068A true CN101603068A (en) | 2009-12-16 |
| CN101603068B CN101603068B (en) | 2012-04-18 |
Family
ID=41468969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009100162503A Expired - Fee Related CN101603068B (en) | 2009-06-23 | 2009-06-23 | Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101603068B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146026A (en) * | 2010-02-10 | 2011-08-10 | 哈尔滨医科大学 | Protosappanin A derivative and preparation method and application thereof |
| CN110553889A (en) * | 2019-09-18 | 2019-12-10 | 宋建华 | he dyeing process of automatic dyeing machine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006007450A2 (en) * | 2004-06-17 | 2006-01-19 | The Texas A & M University System | Detection, evaluation and treatment for advanced prostate cancer |
| WO2006082414A1 (en) * | 2005-02-03 | 2006-08-10 | Cancer Research Technology Limited | Detection and diagnosis of cancer |
-
2009
- 2009-06-23 CN CN2009100162503A patent/CN101603068B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146026A (en) * | 2010-02-10 | 2011-08-10 | 哈尔滨医科大学 | Protosappanin A derivative and preparation method and application thereof |
| CN102146026B (en) * | 2010-02-10 | 2013-04-24 | 哈尔滨医科大学 | Protosappanin A derivative and preparation method and application thereof |
| CN110553889A (en) * | 2019-09-18 | 2019-12-10 | 宋建华 | he dyeing process of automatic dyeing machine |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101603068B (en) | 2012-04-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhong et al. | Following fungal melanin biosynthesis with solid-state NMR: biopolymer molecular structures and possible connections to cell-wall polysaccharides | |
| Scarano et al. | Sustainability: Obtaining natural dyes from waste matrices using the prickly pear peels of Opuntia ficus-indica (L.) Miller | |
| Nadeem et al. | Topological descriptor of 2-dimensional silicon carbons and their applications | |
| CN106928246A (en) | A kind of compound and preparation method thereof and purposes | |
| KR102665505B1 (en) | Fluorescently labeled polysaccharide, manufacturing method and use thereof | |
| Lewandowska et al. | Influence of the static magnetic field and algal extract on the germination of soybean seeds | |
| Pieczykolan et al. | Effects of Supercritical Carbon Dioxide Extraction (SC-CO2) on the content of tiliroside in the extracts from Tilia L. flowers | |
| CN101603068B (en) | Application of hematoxylin in measuring cell multiplication activity and toxic effect of medicine to cells | |
| Wang et al. | Silver nanoparticles enhanced fluorescence for sensitive determination of fluoroquinolones in water solutions | |
| Al-Romaizan et al. | Novel 1, 8-naphthyridine derivatives: design, synthesis and in vitro screening of their cytotoxic activity against MCF7 cell line | |
| Adeel et al. | Sustainable extraction of colourant from harmal seeds (Peganum harmala) for dyeing of bio-mordanted wool fabric | |
| CN103792259B (en) | A kind of method for assessment of Herba Andrographitis antibacterial activity | |
| Wang et al. | Small water clusters stimulate microcystin biosynthesis in cyanobacterial Microcystis aeruginosa | |
| Szukiewicz et al. | Dead zone for hydrogenation of propylene reaction carried out on commercial catalyst pellets | |
| CN104151325A (en) | Fluorescent probe with rhodamine fluorophore as matrix and preparation method of fluorescent probe with rhodamine fluorophore as matrix | |
| Staneva et al. | Photophysical and antibacterial activity of light-activated quaternary eosin Y | |
| CN102250170A (en) | Preparation method and application of two active flavonoid glycosides in okra fruits | |
| CN107375503A (en) | The preparation method of soapberry pericarp activity extract and its antimycotic application | |
| CN104807727B (en) | The detection method of benzophenone compound the amount of migration in a kind of textile | |
| CN102219822B (en) | Ganoderic acid T amide derivative TLTO-A and synthetic method and application thereof | |
| Zhang et al. | Computing topological indices for para-line graphs of anthracene | |
| CN106018688A (en) | Method for estimating toxicity contribution rate of metal nanoparticle ions and nano effect | |
| CN101650364B (en) | Application of hematoxylin in measuring cellulotoxic effect of cell-mediated immunity effect | |
| CN109810113A (en) | A kind of alkaloid compound and the preparation method and application thereof | |
| Juachon et al. | A proposed image-based detection of methamidophos pesticide using peroxyoxalate chemiluminescence system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120418 Termination date: 20120623 |