CN101846626A - Method for detecting activity of abdominal cavity phagocytic cells by flow cytometry - Google Patents
Method for detecting activity of abdominal cavity phagocytic cells by flow cytometry Download PDFInfo
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Abstract
The invention discloses a method for detecting the activity of abdominal cavity phagocytic cells by a flow cytometry, which aims at providing a method for fast, accurately, simply and easily detecting the activity of the phagocytic cells through the quantitative analysis on the number of phagocytic yeasts of the phagocytic cells on the basis of eliminating the influence of unendocytosed fluorescence yeast cells on the analysis results. The method comprises the following steps: preparing beer yeast cell freeze-dried powder; using FITC to mark the beer yeast cells; carrying out incubation on the suspension liquid of the fluorescence marked beer yeast cells and the blood serum of the mice at the room temperature; adding the suspension liquid of the abdominal cavity phagocytic cells into a hole of a culture plate; adding the fluorescence marked beer yeast cells after the incubation with the blood serum of the mice and the identical suspension liquid of the abdominal cavity phagocytic cells into the rest holes in the cell culture plate; carrying out wall adherence incubation on the cell culture plate; and using the flow cytometry to obtain data and carrying out analysis on the data. The method of the invention accurately measures the phagocytic rate and the phagocytic index, and has good repetitiveness.
Description
Technical field
The present invention relates to cell biological experimental technique field, in particular, relate to the method that a kind of characteristics fast and accurately of utilizing flow cytometer detect activity of abdominal cavity phagocytic.
Background technology
The invasion that abdominal cavity phagocytic is resisted pathogen for body plays an important role.Phagocyte belongs to the nospecific immunity cell, can nonspecificly engulf the pathogen that enters in the human body, and old and feeble sick cell participates in antigen recognizing and submission, generates the various kinds of cell factor, plays a significant role in the inherent immunity of body.Therefore, detecting the phagocyte activity is extremely important for the screening of medicine and the exploitation of functional food.And the cytophagous activity of traditional detection mainly contains and engulfs chicken red blood cell and yeast method, because these methods are time-consuming, workload is big, it is bigger influenced by people's subjective factor, the negligible amounts of observation, generally only count 100 phagocytes, so there is certain error.
At present, detect the phagocyte activity with flow cytometer and more and more be subjected to people's attention.Because the flow cytometer detection speed can reach the cell of about 10,000 of per seconds, amounts to several 20,000 cells, improved the accuracy that detects greatly, and provide quick, accurate, a feasible experimental technique for the more experimental design of sample size.Since nineteen eighty-two Steinkamp initiative fluorescent microsphere flow cytometry detection by quantitative phagocyte phagocytic function, up to the present, the technical method that grows up is also a lot, as the method for engulfing the method for fluorescent microsphere and engulfing bacteriums such as fluorescence labeling Escherichia coli, staphylococcus aureus and Much's bacillus.Wherein, engulf in the method for fluorescent microsphere, the fluorescent microsphere particle is homogeneous relatively, after hatching with mice serum, can make its surface bound Ig, good detection effect is arranged, but cost is higher.Be used for detecting the phagocyte activity with some bacteriums such as Escherichia coli and have following shortcoming: the first, cell is less, is easy to be adhered to by the phagocyte surface receptor, influences testing result.The second, utilize these bacteriums of FITC mark, mark rate is lower, therefore can produce certain error in experiment.V.Miliukien
Deng having reported that engulfing the fluorescence labeling brewer's yeast with neutrophil leucocyte in the flow cytometry technology for detection peripheral blood detects its activate the phagocytic capacity, but because it is not removed the fluorescence yeast cells of not engulfing, just utilize forward light and side direction aperture to live phagocyte, because the yeast cells individuality is bigger, number is far more than granulocytic number, therefore, in the cell that flow cytometer obtains, contain a large amount of yeast cells, the granulocyte quantity of obtaining is reduced, simultaneously wherein be mixed with a large amount of yeast cells, also influenced precision of analysis; And in interpretation of result, the number that neutrophil leucocyte is engulfed yeast is not carried out quantitative test, therefore can't calculate granulocytic phagocytic index.
At present, Shang Weiyou utilizes fluorescently-labeled yeast cells to detect the method for activity of abdominal cavity phagocytic as material, because V.Miliukien
Method in have above-mentioned defective, therefore, need on original basis that utilizes flow cytometer to detect the phagocyte activity, explore a cover more accurately, reliable, the easy method of row.
Summary of the invention
The present invention is in order to overcome the existing weak point of utilizing in the flow cytometer detection activity of abdominal cavity phagocytic technology, provide a kind of can the elimination not engulf of the influence of fluorescence yeast cells to analysis result, phagocyte is engulfed the number of yeast and carried out quantitative test, the method for quick, accurate, simple detection phagocyte activity.
The present invention is achieved through the following technical solutions:
A kind of method of utilizing flow cytometer to detect activity of abdominal cavity phagocytic is characterized in that, comprises the steps:
(1) preparation beer yeast cells freeze-dried powder: utilize potato medium culture beer yeast cells, and the beer yeast cells solution that obtains is made the beer yeast cells freeze-dried powder by freeze drying;
(2) utilize FITC mark beer yeast cells: with FITC and dimethyl sulfoxide according to 1mg: the ratio dissolving of 200 μ l, the carbonic acid buffer that be 9.5 with the pH value then, concentration is 0.5M is hybridly prepared into the FITC solution for standby that concentration is 0.1mg/ml; With the beer yeast cells freeze-dried powder that obtains in the step (1) and pH value be 7.2, after concentration is the ratio mixing of PBS damping fluid in 20mg: 1ml of 0.01M, 80 ℃ of deactivation 15min; Centrifugal after the deactivation, abandon supernatant and get precipitation; FITC solution mixing with the sediment that obtains and above-mentioned standby 0.1mg/ml, the room temperature lucifuge is hatched 1h beer yeast cells is carried out fluorescence labeling, wherein, the consumption of the FITC solution of 0.1mg/ml for step (1) in the ratio of the brewer's yeast freeze-dried powder that obtains be 1ml: 20mg; With the solution behind the above-mentioned fluorescence labeling with the pH value be 7.2, concentration is the PBS damping fluid centrifuge washing of 0.01M, and is colourless bright until supernatant; Centrifugal, abandon supernatant and get precipitation and obtain fluorescently-labeled beer yeast cells; Detect the fluorescence labeling rate, adjust the qualified fluorescence labeling beer yeast cells suspension of final fluorescence labeling rate, its fluorescence labeling beer yeast cells concentration is 2 * 10
9Individual/ml preserves standby in 4 ℃ of refrigerators;
(3) be 1: 1 ratio incubated at room 60min with the suspension of the standby fluorescence labeling beer yeast cells that obtains in the step (2) and mice serum according to volume ratio, adjusting fluorescence labeling beer yeast cells concentration with the PBS damping fluid that the pH value is 7.2, concentration is 0.01M is 1 * 10
9Individual/ml;
(4) cytophagous number is at least 1 * 10
6The abdominal cavity phagocytic suspension of individual/ml adds in the hole of culture plate as negative control; With obtain in the step (3) hatch with mice serum after the suspension of fluorescence labeling beer yeast cells and same above-mentioned cytophagous number be at least 1 * 10
6The abdominal cavity phagocytic suspension of individual/ml joins in the Tissue Culture Plate in remaining hole, is that 1: 20 ratio is mixed according to phagocyte and fluorescently-labeled beer yeast cells number ratio; Then Tissue Culture Plate is placed 37 ℃ of adherent 40min of hatching of incubator, remove beer yeast cells unnecessary in the Tissue Culture Plate and do not have adherent abdominal cavity phagocytic with the PBS damping fluid flushing that 4 ℃ of pH values are 7.2, concentration is 0.01M, at last, the 0.5mlpH value that adds 4 ℃ in culture plate each hole except that negative control hole is 7.2, concentration is the PBS damping fluid mixing of 0.01M, the abdominal cavity phagocytic suspension of fluorescence beer yeast cells is engulfed in acquisition, with liquid-transfering gun it is transferred in the streaming pipe, the streaming pipe is placed on ice, treat that the up flow type cell instrument detects;
(5) utilize flow cytometer that the abdominal cavity phagocytic suspension sample of engulfing the fluorescence beer yeast cells that obtains in fluorescence labeling beer yeast cells suspension standby in the step (2) and the step (4) is detected, obtain data and data are analyzed: 1. get the abdominal cavity phagocytic suspension that does not add the fluorescence beer yeast cells in the step (4) and go up sample as negative control, by adjusting FL1 fluorescence channel voltage, make the position at abdominal cavity phagocytic colony peak be positioned at fluorescence voltage transverse axis left side, and abdominal cavity phagocytic is obtained; 2. under the situation that each fluorescence voltage parameter of flow cytometer and threshold value remain unchanged, utilize flow cytometer to record the average fluorescent strength of fluorescently-labeled beer yeast cells standby in the step (2), and with this fluorescence intensity as standard; 3. get the abdominal cavity phagocytic suspension of engulfing the fluorescence beer yeast cells that obtains in the step (4) and go up sample, under the situation that each parameter of flow cytometer remains unchanged, utilize abdominal cavity phagocytic sample in the flow cytometer convection type pipe to carry out cell and obtain; 4. utilize BD CellQuest Pro data analysis software that the abdominal cavity phagocytic data of obtaining are analyzed, draw scatter diagram earlier, by forward light and lateral light, circle is lived abdominal cavity phagocytic colony zone, draws histogram then a M1 is established in negative zone; The average fluorescent strength of step fluorescently-labeled beer yeast cells 2. is made as a standard unit, the cell that 3. step is obtained obtains the result and establishes door according to the multiple of standard unit, from the position of right-hand member to a standard unit of door M1 is M2, at 2 times, 3 times ..., a n-1 times standard unit place establishes door and respectively is M3, M4......Mn.Wherein: M1 represents the abdominal cavity phagocytic number in the M1 door, and promptly negative abdominal cavity phagocytic colony is not for engulfing the abdominal cavity phagocytic sum of beer yeast cells; M2 represents the abdominal cavity phagocytic sum in the M2 door, the abdominal cavity phagocytic number of promptly engulfing 1 yeast cells; Mn represents the abdominal cavity phagocytic number in the Mn door, the abdominal cavity phagocytic number of promptly engulfing n-1 yeast cells;
Determine the value of n according to the number of establishing door, the opposite house inner cell is analyzed, the abdominal cavity phagocytic number and the interior abdominal cavity phagocytic of M1 door that obtain respectively in M1, M2, the M3......Mn door account for total cytophagous percent, calculate cytophagous phagocytic rate and phagocytic index respectively according to following two formula:
Phagocyte accounts for total cytophagous percent in the phagocyte sum=1-M1 door of the phagocyte number of phagocytic rate (%)=engulf beer yeast cells/obtain;
The phagocyte sum of phagocyte sum=[M2+2M3+3M4+......+ (n-1) Mn] of the yeast cells sum that phagocytic index=quilt is engulfed/obtain/obtain.
Utilize the method for potato medium culture beer yeast cells to be: the brewer's yeast bacterial classification to be seeded in inclined-plane potato nutrient culture media to activate, add sterilized water to inclined-plane potato nutrient culture media the inside, gently scrape with oese, the brewer's yeast of inclined-plane potato media surface is suspended from the sterilized water, the brewer's yeast suspension is placed mixing on the vortex vortex mixer, obtain the brewer's yeast bacteria suspension; Draw 0.5ml brewer's yeast bacteria suspension and add dull and stereotyped potato nutrient culture media, after the coating, place 28 ℃ incubator, after 3 days, in each potato plating medium, add sterilized water, scrape with glass bar and get and collect beer yeast cells, after twice of PBS damping fluid 8000rpm * 1min centrifuge washing, 500rpm * 1min is centrifugal, discard precipitation, it is once centrifugal to get upper strata cell suspension 8000rpm * 1min, supernatant discarded, obtain precipitation, be beer yeast cells.After the beer yeast cells suspension that obtains put into-20 ℃ refrigerator and cooled and freeze, insert in the freeze drier freeze drying and make the brewer's yeast freeze-dried powder.
The composition of described potato nutrient culture media is configured by following mass ratio: peeled potatoes: glucose: agar: water=50: 5: 4: 200.
The present invention has following technique effect:
1, method of the present invention is utilized cell data and the easily adherent biological characteristics of phagocyte that flow cytometer obtains, and FITC mark beer yeast cells is near 100% fluorescence labeling rate, eliminated the influence of the fluorescence beer yeast cells do not engulfed, can measure phagocytic rate and phagocytic index accurately analysis result.
2, in the method for the present invention, phagocyte and fluorescence yeast number ratio are 1: 20 in the culture plate, so just guaranteed that yeast cells fully combines with cytophagous, and by flushing several times, removed beer yeast cells and not adherent phagocyte unnecessary in the culture plate, beer yeast cells is to cytophagous interference when having reduced to carry out flow cytometer showed.
3, the present invention makes freeze-dried powder with brewer's yeast by freeze drying, has prolonged the holding time of brewer's yeast, detects the activity of abdominal cavity phagocytic kit for preparation and has good application prospects.And cell size homogeneous has guaranteed the homogeneity of fluorescence intensity and the stability that phagocyte is engulfed the fluorescence yeast.
4, the present invention engulfs the number that the fluorescence intensity behind the beer yeast cells phagocyte engulfs the fluorescence labeling brewer's yeast according to phagocyte and carries out quantitative test, utilize formula among the present invention by simple data computation, phagocytic rate and phagocytic index have been measured comparatively accurately, provide a kind of good reproducibility, the method for quick, accurate, simple detection phagocyte activity.
5, utilize in the method for the present invention brewer's yeast as engulfing particle, utilize FITC to carry out fluorescence labeling, have higher mark rate.
Description of drawings
Fig. 1 is the scatter diagram of the beer yeast cells that is not labeled;
Fig. 2 is not by the histogram of fluorescently-labeled beer yeast cells;
Fig. 3 is the histogram of the fluorescently-labeled brewer's yeast of FITC;
Fig. 4 is not for engulfing the abdominal cavity phagocytic streaming histogram of yeast
Fig. 5 is for measuring the average fluorescent strength of fluorescence labeling brewer's yeast;
Fig. 6 is a scatter diagram of engulfing the abdominal cavity phagocytic of fluorescence labeling brewer's yeast;
Fig. 7 is the streaming histogram that abdominal cavity phagocytic is engulfed the fluorescence labeling brewer's yeast.
Embodiment
Embodiment 1
(1) preparation beer yeast cells freeze-dried powder:
The brewer's yeast bacterial classification is seeded in inclined-plane potato nutrient culture media to be activated, add sterilized water 3ml to inclined-plane potato nutrient culture media the inside, gently scrape with oese, beer yeast cells on the inclined-plane potato nutrient culture media is suspended in the sterilized water, the brewer's yeast suspension is placed mixing on the vortex vortex mixer, obtain the brewer's yeast bacteria suspension.Draw 0.5ml brewer's yeast bacteria suspension and add on the potato culture medium flat plate, after the coating, place 28 ℃ incubator, after 3 days, on each potato culture medium flat plate, add the about 0.1-0.2ml of sterilized water, scrape with glass bar and get and collect beer yeast cells, after twice of the usefulness PBS damping fluid 8000rpm * 1mi n centrifuge washing, 500rpm * 1min is centrifugal, discard precipitation, it is once centrifugal to get upper strata cell suspension 8000rpm * 1min, supernatant discarded, obtain precipitation, be beer yeast cells.After the beer yeast cells solution that obtains put into-20 ℃ refrigerator and cooled and freeze, insert in the freeze drier freeze drying and make the brewer's yeast freeze-dried powder.The composition of described potato nutrient culture media is: peeled potatoes 200g, glucose 20g, agar 16g, water 1000ml.
(2) preparation of mice serum: prepare mice serum as opsonin with kunming mice.Extract eyeball of mouse with tweezers and get blood, room temperature leaves standstill 30min in centrifuge tube, puts 2h at 4 ℃ of refrigerators.Centrifugal 30min under 4 ℃, 1000g centrifugal force draws upper serum subsequently, after the packing, preserves in-20 ℃ of refrigerators.
(3) with F ITC fluorescence labeling beer yeast cells:
1mg FITC is mixedly configured into the FITC solution for standby that concentration is 0.1mg/ml with the carbonic acid buffer that 200 μ l dimethyl sulfoxides dissolving back and pH value are 9.5, concentration is 0.5M.The 120mg brewer's yeast freeze-dried powder that obtains in the step (1) and pH value are 7.2, after concentration is the 6ml PBS damping fluid mixing of 0.01M, put into 80 ℃ of constant water bath box heating deactivation in 15 minutes.The centrifugal 2min of 8000rps abandons the supernatant taking precipitate after the deactivation, and with the FITC solution mixing of the above-mentioned standby 0.1mg/ml of the sediment that obtains and 6ml, the room temperature lucifuge is hatched 1h beer yeast cells is carried out fluorescence labeling.Solution behind the above-mentioned fluorescence labeling is with the pH value is 7.2, concentration is 0.01M PBS damping fluid centrifuge washing 4 times, colourless bright until supernatant.The centrifugal supernatant of abandoning is got precipitation and is obtained fluorescently-labeled beer yeast cells.
Utilize flow cytometer to detect the mark rate of fluorescence labeling yeast:
At first utilize flow cytometer to detect unlabelled beer yeast cells suspension: the PBS damping fluid mixing that a little beer yeast cells freeze-dried powder that step (1) is obtained and pH value are 7.2, concentration is 0.01M is made does not have the beer yeast cells of mark suspension.The beer yeast cells suspension that last sample is not labeled, Fig. 1 is the scatter diagram of the beer yeast cells that is not labeled, analyzing circle R1 zone firmly is beer yeast cells colony, adjust the fluorescence voltage channel, make beer yeast cells colony be in the left side of streaming histogram, and establish negative control door N1, as shown in Figure 2, wherein the negative yeast cells number in the N1 door is 9986, and the ratio that accounts for is 99.86%.
Getting a little fluorescently-labeled beer yeast cells suspension then utilizes flow cytometer to detect the fluorescence labeling rate: it is 7.2 that a little above-mentioned fluorescently-labeled beer yeast cells that obtains is added the pH value, concentration is that the PBS damping fluid mixing of 0.01M is made fluorescently-labeled beer yeast cells suspension, the fluorescently-labeled beer yeast cells suspension of last sample detects, as shown in Figure 3,10000 yeast cells have been obtained altogether, the door of N1 among Fig. 2 is copied among Fig. 3, the right side area of N1 is established a N2 in Fig. 3 then, positive cell number behind the fluorescence labeling among the N2 is 9997, the ratio that accounts for is 99.97%, the fluorescence labeling rate of beer yeast cells is the percent that the interior beer yeast cells number of N2 door accounts for the beer yeast cells sum in R1 zone in Fig. 1 centre circle, be 99.97%, the fluorescence labeling rate is qualified greater than 99.9% explanation mark.
The fluorescently-labeled beer yeast cells that the mark that obtains is qualified adds that the pH value is 7.2, concentration is the PBS damping fluid mixing of 0.01M, and the concentration of adjusting final fluorescence labeling beer yeast cells is 2 * 10
9Individual/ml, the suspension that obtains the fluorescence labeling beer yeast cells is preserved standby in 4 ℃ of refrigerators.
(4) with the mice serum that obtains in the suspension of the standby fluorescence labeling beer yeast cells that obtains in the step (3) and the step (2) be 1: 1 ratio incubated at room 60min according to volume ratio, with the pH value be 7.2, the PBS damping fluid adjustment of 0.01M, it is final that to adjust the fluorescently-labeled beer yeast cells concentration that obtains be 1 * 10
9Individual/ml.
(5) preparation of abdominal cavity phagocytic suspension: experiment is a few days ago injected the meat soup of 1ml to mouse peritoneal.During experiment, disconnected neck is put to death mouse, with syringe to mouse peritoneal injection pH value be 7.2, concentration is the PBS damping fluid 4ml of 0.01M, softly mouse web portion is cut off the abdominal cavity, draw peritoneal fluid 2ml in centrifuge tube, with the pH value be 7.2, the washing of the PBS damping fluid of 0.01M, centrifugally get precipitation after once, add the pH value and be 7.2, concentration is the PBS damping fluid 0.5ml suspension abdominal cavity phagocytic of 0.01M, behind the mixing, adjusting phagocyte concentration is 2 * 10
6Individual/ml, obtain the abdominal cavity phagocytic suspension.
(6) the abdominal cavity phagocytic suspension of preparation in the step (5) is joined in the hole of Tissue Culture Plate as negative control.The abdominal cavity phagocytic suspension of preparation in the suspension of that obtain in the step (4) and fluorescence labeling beer yeast cells after mice serum is hatched and the step (5) being joined in remaining hole of Tissue Culture Plate according to phagocyte and fluorescently-labeled beer yeast cells number ratio is that 1: 20 ratio is mixed, then Tissue Culture Plate is placed 37 ℃ of adherent 40min of hatching of incubator, discard liquid in the hole, adding 4 ℃ of pH values in Tissue Culture Plate each hole except that negative control hole is 7.2, concentration is the PBS damping fluid 1ml of 0.01M, rock flushing gently once, eccysis is removed beer yeast cells unnecessary in the culture plate and is not had adherent abdominal cavity phagocytic, the 0.5mlpH value that adds 4 ℃ then in culture plate each hole except that negative control hole is 7.2, concentration is the PBS damping fluid suction piping and druming of 0.01M, mixing, the abdominal cavity phagocytic suspension of fluorescence beer yeast cells is engulfed in acquisition, with liquid-transfering gun it is transferred in the streaming pipe, the streaming pipe is placed on ice, treat that the up flow type cell instrument detects.
(7) utilize flow cytometer that the abdominal cavity phagocytic suspension of engulfing the fluorescence beer yeast cells and the adherent abdominal cavity phagocytic suspension sample that does not add the fluorescence yeast of hatching that obtains in standby fluorescence labeling beer yeast cells suspension in the step (3) and the step (6) detected, every increment originally obtains 10,000 cell, utilize BD CellQuest Pro to obtain cell and the data of obtaining are analyzed: 1. get in the step (6) the adherent abdominal cavity phagocytic suspension that does not add the fluorescence beer yeast cells of hatching and go up sample as negative control, by adjusting FL1 fluorescence channel voltage, make the position at abdominal cavity phagocytic colony peak be positioned at fluorescence voltage transverse axis left side, if negative control door M1, obtain as shown in Figure 4, and to abdominal cavity phagocytic.2. under the situation that each fluorescence voltage parameter of flow cytometer and threshold value remain unchanged, utilize flow cytometer to record the average fluorescent strength of the fluorescently-labeled beer yeast cells in the step (3), and with this fluorescence intensity as standard, as shown in Figure 5, recording the beer yeast cells fluorescence intensity is 2603.31.3. the abdominal cavity phagocytic suspension of getting in the streaming pipe that obtains in the step (6) of engulfing the fluorescence beer yeast cells is gone up sample, under the situation that each parameter of flow cytometer remains unchanged, utilize abdominal cavity phagocytic sample in the flow cytometer convection type pipe to carry out cell and obtain.4. utilize BDCellQuest Pro data analysis software that the data result that obtains is analyzed, draw scatter diagram earlier, as shown in Figure 6, utilize flow cytometer, peritoneal macrophage zone circle is lived, be made as the R2 zone by forward light and lateral light; Then R2 zone inner cell is analyzed, draw histogram then, as shown in Figure 7,1. the negative control door M1 in is duplicated as the negative control door among Fig. 7, and be made as a standard unit according to the average fluorescent strength 2603.31 of the fluorescence labeling yeast that obtains in 2., and establish door according to the multiple of standard unit, be M2 from the position of right-hand member to a standard unit of door M1, establish door at 2 times, 3 times, 4 times standard unit places and respectively be M3, M4, M5, as shown in Figure 7.Wherein: M1 represents the abdominal cavity phagocytic number in the M1 door, and promptly negative abdominal cavity phagocytic colony is not for engulfing the abdominal cavity phagocytic sum of beer yeast cells; M2 represents the abdominal cavity phagocytic sum in the M2 door, the abdominal cavity phagocytic number of promptly engulfing 1 yeast cells; M3, M4, M5 represent the phagocyte of engulfing 1,2,3,4 fluorescence yeast respectively.Utilize BD CellQuest Pro data analysis software that the data of obtaining are added up, obtain in M2, M3, M4, the M5 door respectively and in total abdominal cavity phagocytic number and the M1 door phagocyte account for total cytophagous percent.Wherein to account for total cytophagous percent be 81.64 to M1 door inner cell; Total abdominal cavity phagocytic number is 7935; M2, M3, M4, M5 door inner cell number are respectively 665,246,236,333.
(8) calculate phagocytic rate and phagocytic index:
Abdominal cavity phagocytic accounts for the percent=1-81.64%=18.36% of total abdominal cavity phagocytic in the phagocyte sum=1-M1 door of the phagocyte number of phagocytic rate (%)=engulf beer yeast cells/obtain
Yeast cells sum/phagocyte sum that phagocytic index=quilt is engulfed=[M2+2M3+3M4+......+ (n-1) Mn]/phagocyte sum=(665+246 * 2+236 * 3+333 * 4)/7935=0.26
Method of the present invention is with cell data and the easily adherent biological characteristics of phagocyte that flow cytometer obtained, and FITC mark beer yeast cells is near 100% fluorescence labeling rate, eliminated the influence to analysis result of the fluorescence beer yeast cells do not engulfed.This method can reduce the error that flow cytometer detects the abdominal cavity phagocytic activate the phagocytic capacity, at can not measure the defective of engulfing number accurately in the detection method of being set up in the past, the present invention engulfs the number that the fluorescence intensity behind the yeast cells phagocyte engulfs the fluorescence labeling brewer's yeast according to phagocyte and carries out quantitative test, utilize formula among the present invention by simple data computation, phagocytic rate and phagocytic index have been measured comparatively accurately, provide a kind of good reproducibility, the method for quick, accurate, simple detection phagocyte activity.
Claims (3)
1. a method of utilizing flow cytometer to detect activity of abdominal cavity phagocytic is characterized in that, comprises the steps:
(1) preparation beer yeast cells freeze-dried powder: utilize potato medium culture beer yeast cells, and the beer yeast cells solution that obtains is made the beer yeast cells freeze-dried powder by freeze drying;
(2) utilize FITC mark beer yeast cells: with FITC and dimethyl sulfoxide according to 1mg: the ratio dissolving of 200 μ l, the carbonic acid buffer that be 9.5 with the pH value then, concentration is 0.5M is hybridly prepared into the FITC solution for standby that concentration is 0.1mg/ml; With the beer yeast cells freeze-dried powder that obtains in the step (1) and pH value be 7.2, after concentration is the ratio mixing of PBS damping fluid in 20mg: 1ml of 0.01M, 80 ℃ of deactivation 15min; Centrifugal after the deactivation, abandon supernatant and get precipitation; FITC solution mixing with the sediment that obtains and above-mentioned standby 0.1mg/ml, the room temperature lucifuge is hatched 1h beer yeast cells is carried out fluorescence labeling, wherein, the consumption of the FITC solution of 0.1mg/ml for step (1) in the ratio of the brewer's yeast freeze-dried powder that obtains be 1ml: 20mg; With the solution behind the above-mentioned fluorescence labeling with the pH value be 7.2, concentration is the PBS damping fluid centrifuge washing of 0.01M, and is colourless bright until supernatant; Centrifugal, abandon supernatant and get precipitation and obtain fluorescently-labeled beer yeast cells; Detect the fluorescence labeling rate, adjust the qualified fluorescence labeling beer yeast cells suspension of final fluorescence labeling rate, its fluorescence labeling beer yeast cells concentration is 2 * 10
9Individual/ml preserves standby in 4 ℃ of refrigerators;
(3) be 1: 1 ratio incubated at room 60min with the suspension of the standby fluorescence labeling beer yeast cells that obtains in the step (2) and mice serum according to volume ratio, adjusting fluorescence labeling beer yeast cells concentration with the PBS damping fluid that the pH value is 7.2, concentration is 0.01M is 1 * 10
9Individual/ml;
(4) cytophagous number is at least 1 * 10
6The abdominal cavity phagocytic suspension of individual/ml adds in the hole of culture plate as negative control; With obtain in the step (3) hatch with mice serum after the suspension of fluorescence labeling beer yeast cells and same above-mentioned cytophagous number be at least 1 * 10
6The abdominal cavity phagocytic suspension of individual/ml joins in the Tissue Culture Plate in remaining hole, is that 1: 20 ratio is mixed according to phagocyte and fluorescently-labeled beer yeast cells number ratio; Then Tissue Culture Plate is placed 37 ℃ of adherent 40min of hatching of incubator, remove beer yeast cells unnecessary in the Tissue Culture Plate and do not have adherent abdominal cavity phagocytic with the PBS damping fluid flushing that 4 ℃ of pH values are 7.2, concentration is 0.01M, at last, the 0.5mlpH value that adds 4 ℃ in culture plate each hole except that negative control hole is 7.2, concentration is the PBS damping fluid mixing of 0.01M, the abdominal cavity phagocytic suspension of fluorescence beer yeast cells is engulfed in acquisition, with liquid-transfering gun it is transferred in the streaming pipe, the streaming pipe is placed on ice, treat that the up flow type cell instrument detects;
(5) utilize flow cytometer that the abdominal cavity phagocytic suspension sample of engulfing the fluorescence beer yeast cells that obtains in fluorescence labeling beer yeast cells suspension standby in the step (2) and the step (4) is detected, obtain data and data are analyzed: 1. get the abdominal cavity phagocytic suspension that does not add the fluorescence beer yeast cells in the step (4) and go up sample as negative control, by adjusting FL1 fluorescence channel voltage, make the position at abdominal cavity phagocytic colony peak be positioned at fluorescence voltage transverse axis left side, and abdominal cavity phagocytic is obtained; 2. under the situation that each fluorescence voltage parameter of flow cytometer and threshold value remain unchanged, utilize flow cytometer to record the average fluorescent strength of fluorescently-labeled beer yeast cells standby in the step (2), and with this fluorescence intensity as standard; 3. get the abdominal cavity phagocytic suspension of engulfing the fluorescence beer yeast cells that obtains in the step (4) and go up sample, under the situation that each parameter of flow cytometer remains unchanged, utilize abdominal cavity phagocytic sample in the flow cytometer convection type pipe to carry out cell and obtain; 4. utilize BD CellQuest Pro data analysis software that the abdominal cavity phagocytic data of obtaining are analyzed, draw scatter diagram earlier, by forward light and lateral light, circle is lived abdominal cavity phagocytic colony zone, draws histogram then a M1 is established in negative zone; The average fluorescent strength of step fluorescently-labeled beer yeast cells 2. is made as a standard unit, the cell that 3. step is obtained obtains the result and establishes door according to the multiple of standard unit, from the position of right-hand member to a standard unit of door M1 is M2, at 2 times, 3 times ..., n-1 times standard unit place establishes door and respectively is M3, M4......Mn, wherein: M1 represents the abdominal cavity phagocytic number in the M1 door, be negative abdominal cavity phagocytic colony, for not engulfing the abdominal cavity phagocytic sum of beer yeast cells; M2 represents the abdominal cavity phagocytic sum in the M2 door, the abdominal cavity phagocytic number of promptly engulfing 1 yeast cells; Mn represents the abdominal cavity phagocytic number in the Mn door, the abdominal cavity phagocytic number of promptly engulfing n-1 yeast cells;
Determine the value of n according to the number of establishing door, the opposite house inner cell is analyzed, the abdominal cavity phagocytic number and the interior abdominal cavity phagocytic of M1 door that obtain respectively in M1, M2, the M3......Mn door account for total cytophagous percent, calculate cytophagous phagocytic rate and phagocytic index respectively according to following two formula:
Phagocyte accounts for total cytophagous percent in the phagocyte sum=1-M1 door of the phagocyte number of phagocytic rate (%)=engulf beer yeast cells/obtain;
The phagocyte sum of phagocyte sum=[M2+2M3+3M4+......+ (n-1) Mn] of the yeast cells sum that phagocytic index=quilt is engulfed/obtain/obtain.
2. the method for utilizing flow cytometer to detect activity of abdominal cavity phagocytic according to claim 1, it is characterized in that, utilize the method for potato medium culture beer yeast cells to be: the brewer's yeast bacterial classification to be seeded in inclined-plane potato nutrient culture media to activate, add sterilized water to inclined-plane potato nutrient culture media the inside, gently scrape with oese, the brewer's yeast of inclined-plane potato media surface is suspended from the sterilized water, the brewer's yeast suspension is placed mixing on the vortex vortex mixer, obtain the brewer's yeast bacteria suspension; Draw 0.5ml brewer's yeast bacteria suspension and add dull and stereotyped potato nutrient culture media, after the coating, place 28 ℃ incubator, after 3 days, in each potato plating medium, add sterilized water, scrape with glass bar and get and collect beer yeast cells, after twice of PBS damping fluid 8000rpm * 1min centrifuge washing, 500rpm * 1min is centrifugal, discard precipitation, it is once centrifugal to get upper strata cell suspension 8000rpm * 1min, supernatant discarded, obtain precipitation, be beer yeast cells; After the beer yeast cells suspension that obtains put into-20 ℃ refrigerator and cooled and freeze, insert in the freeze drier freeze drying and make the brewer's yeast freeze-dried powder.
3. the method for utilizing flow cytometer to detect activity of abdominal cavity phagocytic according to claim 1, it is characterized in that the composition of described potato nutrient culture media is configured by following mass ratio: peeled potatoes: glucose: agar: water=50: 5: 4: 200.
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