CN110196219B - Chicken spleen CD8 detection based on flow cytometry+Method for specific killing of T cells - Google Patents
Chicken spleen CD8 detection based on flow cytometry+Method for specific killing of T cells Download PDFInfo
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- C12N5/0602—Vertebrate cells
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Abstract
The invention relates to a method for detecting chicken spleen CD8 based on flow cytometry+A method of T cell specific killing comprising the steps of: (1) culturing bacteria such as Salmonella pullorum S06004 and S06004 deltaspiC(ii) a (2) Immunizing and sampling the chicken; (3) preparing target cell-macrophage; (4) effector cell-chicken spleen CD8+Preparation of T cells: comprises the steps of preparing chicken spleen lymphocyte suspension and sorting chicken spleen CD8 by magnetic beads+A step of T cells; (5) CTL specifically kills macrophages. Compared with the prior art, the invention has the following beneficial effects: the macrophage (target cell) obtained by the invention has higher purity, and the chicken spleen CD8+The T cell sorting effect is obvious, and the separation time is short; the salmonella host cell-macrophage is taken as a target cell, so that the removing capability of the host on the salmonella can be more accurately reflected; flow cytometry was used for chicken spleen CD8+Specific killing effect of T cells, and capability of detecting chicken spleen CD8 at single cell level+CTL effect of T cells.
Description
Technical Field
The invention relates to a method for detecting chicken spleen CD8 based on flow cytometry+T cell specific killing method.
Background
The cellular immune response is mediated by T lymphocytes, which is beneficial to promoting the generation of body antibodies and also beneficial to eliminating pathogenic microorganisms (such as bacteria, viruses and the like) parasitizing in cells. In addition, cellular immune responses are also important in poultry immune surveillance and in anti-tumor (e.g., Marek's disease) immune processes. After poultry is infected with pathogenic microorganisms, T cells play a crucial role in the immune response process by producing proinflammatory cytokines, modulating local and systemic immune responses, activating macrophages, and eliminating intracellular bacteria, among other things. Among them, cytotoxic T Cells (CTLs) have lytic activity, play a key role in the recognition and elimination of abnormal cells such as tumors and pathogen-infected cells, and kill target cells mainly through two pathways of perforin/granzyme and death receptor, and thus, the determination of cytotoxic activity is of great significance in both basic theoretical research and production practice.
Currently, there are many methods for detecting cytotoxicity: MTT reduction method, LDH release method, reporter gene transfection method, ELISPOT assay, and radioactive chromium: (51Cr) release assay, etc. Among them, the most popular test for assessing cytotoxicity is radiochromium (b) ((b))51Cr) release assay, which is considered to be a "gold standard" for detecting cell-mediated cytotoxicity, has the advantages of high reproducibility and relative ease of implementation, but has low sensitivity, strong radioactivity, detects lysis of cells at the global level, rather than at the single cell level, and provides only semi-quantitative data. Therefore, there is a need to establish a safe, quantitative, high-throughput method for detecting cytotoxicity.
With the popularization of fluorometric instruments and the discovery of new fluorescent markers, fluorometry will likely replace traditional fluorescent markers51Cr Release method, the disadvantages explained aboveThe development of flow cytometry is facilitated by traps. Flow cytometry cytotoxicity assays have the advantages of being radioactivity free, detecting cytotoxicity at the single cell level, and assessing all stages of the cytotoxic process. At present, the chicken spleen CD8 detected based on flow cytometry is not available+Reports on T cell specific killing.
Disclosure of Invention
The object of the invention is directed to radioactive chromium (A), (B), (C) and C)51Cr) release has the defects of low sensitivity, strong radioactivity and the like, and establishes a more updated and reliable method capable of detecting cytotoxicity at single cell level (namely a method for detecting chicken spleen CD8 based on flow cytometry+A method of T cell specific killing).
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: chicken spleen CD8 detection based on flow cytometry+A method for specific killing of T cells, characterized in that chicken CD8 can be detected at the single cell level+Specific killing of T cells comprising the steps of:
(1) culturing bacteria, wherein the bacteria are salmonella pullorum S06004 and S06004 delta spiC;
(2) immunizing and sampling the chicken;
(3) the preparation of the target cell-macrophage, which takes the primary macrophage as the target cell for the first time, is closer to the natural state of an organism and provides a more accurate result;
(4) effector cell-chicken spleen CD8+Preparation of T cells: comprises the steps of preparing chicken spleen lymphocyte suspension and sorting chicken spleen CD8 by magnetic beads+A step of T cells;
(5) CTL specifically kills macrophages, and specific killing capacity at a single cell level can be reflected by flow detection of apoptosis.
The step (1) is specifically as follows: selecting single salmonella S06004 and S06004 delta spiC colonies, inoculating the single salmonella S06004 and S06004 delta spiC colonies in an LB liquid culture medium, culturing to a logarithmic growth phase, and adjusting the bacterial concentration.
The step (2) is specifically as follows: 3 day old chicks were randomly divided into 2 groups and given intramuscular injections of immune PBS and S06004. delta. spiC, respectively.
The step (3) is specifically as follows: separating peripheral blood lymphocytes of the chicken by using lymphocyte separating medium, carrying out adherent culture to obtain macrophages, marking part of adherent macrophages by using a mouse anti-chicken macrophage antibody, carrying out purity determination, inoculating the rest macrophages to a 96-well plate, and culturing overnight. The host cell (macrophage) of the salmonella is taken as the target cell of the CTL for the first time, and the clearance capability of the host to the salmonella intracellularis can be more accurately reflected.
The preparation method of the chicken spleen lymphocyte suspension comprises the following steps: grinding and filtering chicken spleen to obtain lymphocyte suspension, centrifuging at low speed for 2 times to remove part of erythrocytes, and separating with lymphocyte separating medium to obtain chicken lymphocytes.
Magnetic bead sorting chicken spleen CD8+The T cell steps are specifically: labeling the prepared chicken lymphocytes with CD8 alpha-APC and anti-mouse IgG magnetic beads as primary antibody and secondary antibody respectively, and separating by LS column to obtain chicken spleen CD8+T cells, a portion of chicken spleen CD8+The purity of the T cells was determined.
The step (5) is specifically as follows: adding the bacteria S06004 into overnight cultured macrophages, centrifuging, culturing for 1 h, adding a culture medium containing 100 mug/mL gentamicin, and culturing for 1 h to kill bacteria outside the macrophages; replacing culture medium containing gentamicin of 10 mug/mL to inhibit proliferation of bacteria outside macrophages, continuing culturing for 5 h, and adding chicken spleen CD8+T cells are cultured for 24 h together, the cells are collected, the apoptosis of target cells (namely macrophages) is detected, and the chicken CD8 can be evaluated at the single cell level+Specific killing of T cells.
Compared with the prior art, the invention has the following beneficial effects:
1. the purity of the target cells (namely macrophages) obtained by adherent culture is higher, and compared with other CTL detection methods, the method takes primary cells as the target cells, is closer to the natural state of an organism and provides more accurate results;
2. the macrophage is a salmonella host cell and is used as a target cell of CTL for the first time, and the clearance of the host on salmonella can be more accurately reflected.
3. The invention adopts the magnetic bead to sort chicken spleen CD8+The T cells have the advantages of obvious sorting effect, short separation time, high cell activity and the like.
4. The invention applies flow cytometry to the chicken spleen CD8 for the first time+T cell specific killing effect, and the CD8 in the spleen of the immunized chicken is verified+T cells have a strong CTL killing effect. Flow cytometry can detect CTL effects at the single cell level and has higher sensitivity (because it detects early markers (apoptosis)) than other methods of detecting CTL specific killing ability.
Drawings
FIG. 1 identification of macrophage purity by adherent culture:
(A) preattached negative control unlabeled group; (B) purity of macrophages before adherence; (C) post-adherent negative control unlabeled group; (D) purity of macrophage after adherence;
FIG. 2 magnetic bead sorting of chicken spleen CD8+And (3) identifying the purity of the T cells:
(A) negative control unlabeled group; (B) CD8 before sorting+Purity of the T cells; (C) sorted CD8+Purity of the T cells;
fig. 3 flow cytometry analysis of CTL specific killing ability:
(A) non-immunized group chicken spleen CD8+T cell induced target cell apoptosis; (B) immune group chicken spleen CD8+T cell induced target cell apoptosis; (C) ratio of each cell after CTL-specific killing.
Detailed Description
The invention is described in further detail below with reference to the figures and specific examples.
1. Material
1.1 Experimental animals
1.2 strains of Bacillus subtilis
Salmonella pullorum S06004 (Nal)R) Separated preservation by this laboratory, gather people: gunn faithful, collecting people contact information 0514-: xuzhou, Jiangsu, with a collection time of 3 months in 2006; salmonella pullorum mutant strain S06004 delta spiC mutant strain constructed and stored in the same room (construction of Salmonella pullorum S06004 delta spiC mutant strain for male Liu and preliminary research on immunobiological characteristics thereof [ D]Yangzhou university, 2012).
2. Method of producing a composite material
2.1 bacterial culture
And (3) selecting S06004 and S06004 delta spiC single colonies, inoculating the single colonies into 4 mL LB liquid culture medium, performing shake culture at 37 ℃ and 180 r/min for 12 h by using a shaking table, inoculating the bacterial liquid into a conical flask in a volume of 1:500 for amplification culture, performing shake culture for 14 h, taking out, and performing standing culture for 3-4 h. The cells were washed 3 times with sterile PBS to adjust the concentration of the bacteria (S06004, S06004. delta. spiC).
2.2 immunization and sampling of chickens
3 day old chicks were randomly divided into 2 groups and immunized with PBS and S06004. delta. spiC (1X 10) by intramuscular injection, respectively7 CFU/plume), 100 μ L/plume.
2.3 preparation of target cell-macrophage
Diluting peripheral blood of the chicken collected in an anticoagulation tube by using DPBS (platelet-derived plasma) in equal volume; adding diluted peripheral blood into Histopaque-1083 with the same volume, centrifuging at 20 deg.C and 2000 r/min for 20 min, and collecting intermediate white mist layer cells; diluting the white haze layer cells obtained by using the equal volume of DPBS, uniformly mixing the white haze layer cells, adding the white haze layer cells into the equal volume of lymphocyte separation liquid, and separating again under the same condition to ensure that high-purity white blood cells are obtained; the obtained cells were resuspended in DPBS, washed twice, and the cell concentration was adjusted to 1X 10 with RPMI 1640 medium containing 5% fetal bovine serum, 5% chicken serum, 100U/mL cyan, and streptomycin7Adding 10 mL of the cells into a cell culture dish; 5% CO at 41 deg.C2The incubator is kept static for culture, the culture medium and the cells which are not attached to the wall are discarded after 24 hours, and the fresh culture medium is replaced for continuous culture for 24 hours; then, the culture medium and the suspension cells are discarded, and the cells attached to the wall in the culture dish are the macrophages.
The obtained macrophages were washed twice with PBS; digesting with EDTA-free pancreatin for 3 min, adding chicken extract containing 5%Complete RPMI 1640 of serum, 5% FBS, 100U/mL cyan, streptomycin, 0.1% β -mercaptoethanol, and 25% HEPES stopped the pancreatin reaction; blowing and beating the adherent cells, and centrifuging at the temperature of 4 ℃ at 1000 r/min for 10 min; discarding the supernatant; taking a part of cells, marking the cells with Mouse Anti-Chicken macrophage antibody (Mouse Anti-Chicken monoclonal/macro-PE) (the marked cells are only used for purity determination), and performing purity determination by a FACSAria SORP flow cytometer; the remaining macrophages (for subsequent experiments: 2.5 CTL-specific killing of macrophages) were resuspended in complete RPMI 1640 medium and the cell concentration was adjusted to 5X 105Per mL; inoculating to 96-well plate (100 μ L/well) at 37 deg.C and 5% CO2The culture was carried out overnight.
2.4 Effector-spleen CD8+Preparation of T cells
2.4.1 preparation of Chicken spleen lymphocyte suspension
After the chickens were sacrificed by cardiac injection of air, the spleens were taken and placed in 15 mL centrifuge tubes containing DPBS; washing with DPBS for 2 times, transferring the spleen to a petri dish, mincing with sterile scissors and transferring to a Gentle MACS C tube containing 6 mL of DPBS, and grinding by a Gentle MACS fully automatic tissue processor; filtering with a filter of 70 mu m, and taking filtrate; centrifuging at 600 r/min for 5 min, sucking supernatant into a new centrifuge tube, and repeating for 2 times to remove most of erythrocytes; slowly adding the cell suspension into the lymphocyte separation liquid with the same volume, and centrifuging for 20 min at the temperature of 20 ℃ by a horizontal rotor 1800 r/min; sucking the middle white spray layer lymphocytes, supplementing 6 mL of complete RPMI 1640 medium (containing 10% FBS, 100U/mL of cyan and streptomycin) at 4 ℃, and centrifuging at 1000 r/min for 10 min; the supernatant was discarded to obtain lymphocytes.
2.4.2 magnetic bead sorting of Chicken spleen CD8+T cells
Washing the prepared lymphocytes for 2 times by using a precooled MACS buffer, resuspending the cells, adding CD8 alpha-APC for marking, and keeping the cells away from light for 30 min at 4 ℃; adding 8 mL of precooled MACS buffer, centrifuging at 4 ℃ and 1500 r/min for 10 min to remove the unbound antibody; every 10 th7The cells were resuspended in 100. mu.L of MACS buffer and 20. mu.L of magnetic beads against Mouse IgG (Anti-Mouse IgG 1-Microbeads) were added protected from light at 4 ℃ for 15 min; add 8 mL of pre-mixCentrifuging at 1500 r/min for 10 min at 4 ℃ for cold MACS buffer to remove unbound magnetic beads; resuspend with 500. mu.L of MACS buffer (ca. 10) before magnetic sorting8Individual cells); the LS column was placed on a MACS separator (an ultra strong magnet) and washed with 3 mL of MACS buffer; adding the cell suspension to a column, and magnetically labeling chicken spleen CD8+T cells are adsorbed in the column, while unlabeled cells flow out of the column; washing the column with 3 mL of MACS buffer for 3 times, then removing the LS column from the separator, adding an appropriate amount of MACS buffer to the column, and washing out the cells labeled with the magnetic beads through a piston; resuspend and count with PBS and purity determination by flow cytometry.
2.5 CTL specific killing of macrophages
Macrophages were seeded into 96-well plates at 5X 10 per well4Individual cell, 37 ℃ and 5% CO2Culturing overnight to monolayer cells; 1 h before inoculation of the bacteria S06004, changing a cell culture medium into an RPMI 1640 culture medium containing 5% fetal calf serum, 5% chicken serum and no antibiotics; culturing S06004 to logarithmic growth phase, centrifuging at 4500 r/min for 5 min, discarding supernatant, washing with PBS for 3 times, and infecting macrophage with bacteria S06004 at MOI =10 ratio; centrifuging at 20 deg.C for 10 min at 1000 r/min to make bacteria S06004 fully contact with macrophage; culturing at 37 deg.C for 1 h; discarding the supernatant, washing the macrophages for 3 times by PBS, adding an RPMI 1640 culture medium containing 5% fetal calf serum, 5% chicken serum and 100 mug/mL gentamicin, and culturing at 37 ℃ for 1 h to sufficiently kill bacteria outside the cells; washing with PBS for 3 times, and replacing with RPMI 1640 medium containing 5% fetal calf serum, 5% chicken serum and 10 μ g/mL gentamicin to inhibit proliferation of extracellular bacteria; the culture was continued for 5 h, adding 1X 10 of each well6Chicken spleen CD8+A T cell; 37 ℃ and 5% CO2Co-culturing for 24 h, taking out non-adherent cells, and digesting adherent macrophages by EDTA-free pancreatin; detecting the apoptosis condition of target cells (namely macrophages) according to an operation manual of an apoptosis detection kit, wherein the steps are approximately as follows: the cells were washed twice with 4 ℃ pre-chilled PBS and resuspended in binding buffer, adjusted to a concentration of 1X 107Per mL; taking 100 mu L of cells, adding 5 mu L of Annexin V/Alexa Fluor 488 and 10 mu L of propidium iodide solution; light protection 15min, analyzing by a flow cytometer.
3 results
3.1 macrophage adherence preparation results in peripheral blood
Macrophage prepared by adherence is digested by EDTA-free pancreatin, marked by a mouse anti-chicken macrophage antibody, and the purity is determined by flow cytometry. As can be seen in fig. 1, macrophage purity was 9.76% before nonadherent; the purity of macrophage obtained by adherence reaches 97.7 percent, which shows that the purity of macrophage obtained by adherence is higher, and the method is simple and can be better used for later period experiments.
3.2 spleen CD8+T lymphocyte magnetic bead sorting results
The prepared lymphocytes are marked by a primary antibody and a secondary antibody, magnetic separation is carried out by an LS column, and the purity of the separated cells is measured by flow cytometry. As can be seen in FIG. 2, CD8 was prior to sorting+The proportion of the T lymphocytes is 39.5%, the purity after sorting reaches 93.5%, the sorting effect is obvious, and the separation time is short.
3.3 CTL specific killer macrophage assay results
The results of CTL-specific killing in apoptosis assay are shown in FIG. 3, and it can be seen from FIG. 3A that spleen CD8 was obtained from the non-immunized group of chickens+After the T cells and the target cells are co-cultured for 24 hours, the proportion of live cells of macrophages (target cells) is 86.9 percent, the proportion of apoptotic cells is 5.52 percent, and the proportion of dead cells is 7 percent; as can be seen from FIG. 3B, the spleen CD8 of the immunized group of chickens+After the T cells and the target cells are co-cultured for 24 hours, the proportion of the living cells of the target cells is 67.4 percent, the proportion of the apoptotic cells is 21.7 percent and the proportion of the dead cells is 10.5 percent; the results in FIG. 3C show that the proportion of viable cells of the target cells in the immunized group was significantly reduced compared to the control group (P)<0.05), the proportion of apoptotic cells is remarkably increased (P)<0.05), the proportion of dead cells was also increased, but there was no significant difference, indicating CD8 in chicken spleen after immunization+T cells have a strong CTL killing effect.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. Chicken spleen CD8 detection based on flow cytometry+A method for specific killing of T cells, characterized in that chicken CD8 can be detected at the single cell level+Specific killing of T cells comprising the steps of:
(1) culturing bacteria such as Salmonella pullorum S06004 and S06004 deltaspiC；
(2) Immunizing and sampling the chicken;
(3) the preparation of target cell-macrophage, which takes the separated primary macrophage as the target cell, is closer to the natural state of the body, and provides more accurate results:
separating peripheral blood lymphocytes of the chicken by using lymphocyte separating medium, carrying out adherent culture to obtain macrophages, marking part of adherent macrophages by using a mouse anti-chicken macrophage antibody, carrying out purity determination, inoculating the rest macrophages to a 96-well plate, and culturing overnight;
(4) effector cell-chicken spleen CD8+Preparation of T cells: comprises the steps of preparing chicken spleen lymphocyte suspension and magnetic bead sorting chicken spleen CD8+A step of T cells;
magnetic bead sorting chicken spleen CD8+The T cell steps are specifically: labeling chicken lymphocytes with CD8 alpha-APC and anti-mouse IgG magnetic beads as primary antibody and secondary antibody respectively, and separating by LS column to obtain chicken spleen CD8+T cells, a portion of chicken spleen CD8+Performing purity measurement on the T cells; (5) CTL specifically kills macrophages, apoptosis is detected through flow cytometry, and specific killing capacity can be detected at a single cell level.
2. The flow cytometry-based detection of chickens of claim 1Spleen CD8+The method for killing the T cells specifically is characterized in that the step (1) is as follows: selecting Salmonella pullorum S06004, S06004 deltaspiCThe single colony is inoculated in LB liquid culture medium and cultured to logarithmic phase, and the bacterial concentration is adjusted.
3. The flow cytometry-based chicken spleen CD8 detection method according to claim 1+The method for killing the T cells specifically is characterized in that the step (2) specifically comprises the following steps: randomly dividing 3-day-old chicks into 2 groups, and respectively performing intramuscular injection of immune PBS and S06004 deltaspiC。
4. The flow cytometry-based chicken spleen CD8 detection method according to claim 1+The method for killing the T cell specificity is characterized in that the preparation steps of the chicken spleen lymphocyte suspension are as follows: grinding and filtering chicken spleen to obtain lymphocyte suspension, centrifuging at low speed for 2 times to remove part of erythrocytes, and separating with lymphocyte separating medium to obtain chicken lymphocytes.
5. The flow cytometry-based chicken spleen CD8 detection method according to claim 1+The method for the specific killing effect of the T cells is characterized in that the step (5) is specifically as follows: adding the bacteria S06004 into overnight cultured macrophages, centrifuging, culturing for 1 h, adding a culture medium containing 100 mug/mL gentamicin, and culturing for 1 h to kill bacteria outside the macrophages; replacing culture medium containing 10 mug/mL gentamicin to inhibit proliferation of extracellular bacteria, continuing culturing for 5 h, and adding spleen CD8+T cells are cultured for 24 h together, the cells are collected, the apoptosis of target cells is detected, and chicken spleen CD8 is evaluated+T cell specific killing effect.
6. Flow cytometry-based detection of chicken spleen CD8 according to any one of claims 1-5+The method of the T cell specific killing effect can be used for evaluating the immunity effect and the cellular immune response of the salmonella vaccine.
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