SummaryDuring the immune response, B cells stimulated by different antigens can switch to express different heavy chain constant region genes and change the type of immunoglobulin (Ig) molecules produced.LPS stimulates IgG2b and IgG3 class switching in murine spleen B cells. Cytokines can induce class switching of Ig to specific classes, e.g. IL-4 and LPS together activate B cells to induce class switching of IgG1 and IgE in mice and IgG4 and IgE in humans. ELISA can be used to detect the class of antibody secreted in the supernatant of a cell culture, and ELISPOT can be used to detect the number of cells secreting the corresponding class of antibody. Flow cytometry can be used to detect the expression of the appropriate antibody class by individual cells.
Principle
The rationale for the induction and detection of immunoglobulin class switching is that LPS stimulates IgG2b and IgG3 class switching in murine splenic B cells. Cytokines can induce class switching of Ig to specific classes, e.g., IL-4 and LPS together activate B cells to induce class switching of IgG1 and IgE in mice, and IgG4 and IgE in humans. ELISA can be used to detect the class of antibody secreted in cell culture supernatants, and ELISPOT can be used to detect the number of cells secreting the corresponding class of antibody. Flow cytometry can be used to detect the expression of the appropriate antibody class by individual cells.
Operation method
Induction and detection of immunoglobulin class switching
Principle
The rationale for the induction and detection of immunoglobulin class switching is that LPS stimulates IgG2b and IgG3 class switching in murine splenic B cells. Cytokines can induce class switching of Ig to specific classes, e.g., IL-4 and LPS together activate B cells to induce class switching of IgG1 and IgE in mice, and IgG4 and IgE in humans. ELISA can be used to detect the class of antibody secreted in cell culture supernatants, and ELISPOT can be used to detect the number of cells secreting the corresponding class of antibody. Flow cytometry can be used to detect the expression of the appropriate antibody class by individual cells.
Materials and Instruments
Reagents:
(1) LPS.
(2) Recombinant IL-4.
(3) PBS containing 0.5% BSA with 0.02% sodium azide.
(4) 4% paraformaldehyde.
(5) Anti-mouse or human FcyRIIb monoclonal antibody.
(6) Fluorescently labeled anti-Ig and anti-surface molecule antibodies.
(7) Ig ELISA test kit, Ig ELISPOT test kit.
(8) Antigens related to B cell activation.
(9) RPMI 1640 culture medium containing 10% FCS.
(10) Staining buffer (PBS with 5% BSA).
(11) membrane-breaking solution (1xPBS + 0.1% bovine serum albumin + 0.05% sodium azide + 0.1% saponin).
Apparatus: 48-well culture plate, 96-well culture plateMove
The basic process of induction and detection of immunoglobulin class switching can be divided into the following steps:
(1) Selection of cell culture conditions
(1) LPS (40 μg/mL) to induce class switching of IgG2b and IgG3.
(2) LPS (40 μg/mL) + recombinant IL-4 (5-20 ng/mL), to induce class switching of IgG1 and IgE. (3) Anti-CD40 antibody/CD40L (10 μg/mL), to induce class switching of IgG2b and IgG3. (4) Anti-CD40 antibody/CD40L (10 μg/mL) + recombinant IL-21, to induce class switching of IgG1 and IgG3. (4) Anti-CD40 antibody/CD40L (10 μg/mL) + recombinant IL-21 induced class switching of IgG1 and IgG3. (5) Recombinant IL-10 (10 ng/mL) + recombinant TGFβ (2 ng/mL) induced the initial B cells to secrete IgG (mainly IgG1 and IgG3) and IgA (mainly IgA1 and IgA2). (2) Detection of immunoglobulin secreted by B cells by ELISA (1) Cell preparation: isolate human PBMC or mouse splenic single nucleated cells, purify B cells (2) Cell culture: adjust the cell concentration to 2x10/mL with RPMI 1640 culture medium, select suitable culture conditions to stimulate the cells, take 200 μl and add it to a 96-well round-bottomed culture plate, with 3 replicate wells for each condition, at 37 ℃ and 5% CO2, and then add 200 μl to a 96-well round-bottomed culture plate. Incubate the cells at 37 ℃, 5% CO2 incubator for 2~7 days, collect the supernatant, and test the content of different immunoproteins by ELISA.(3) ELISA to detect Ig.(3) ELISPOT to detect the class of immunoglobulin secreted by B cells.(1) Cell Preparation: Separate the single nucleated cells of human PBMC or mouse spleen, and purify the B cells.(2) Coat capture antibody: Dilute the capture antibody in sterile PBS, and take 100 μl of the antibody into the plate and then dilute the antibody into the plate, then take 100 μl of the antibody into the plate. 2) Capture antibody: Dilute the capture antibody with sterile PBS, take 100 μl of the antibody into a PVDF 96-well plate and incubate at 4 ℃ overnight. 3) Closure: Discard the liquid, wash the PVDF 96-well plate with sterile PBS, 350 μl/well/times, discard the liquid, and add the closure solution (PBS containing 1% BSA and 5% sucrose), 200 μl/well, and let it stand for 2 hours at room temperature. 4) Cell culture: Adjust the cell concentration to 2x2 by using RPMI 1640 culture medium. 4) Cell culture: Adjust the cell concentration to 2x10/mL with RPMI 1640 culture medium, select suitable culture conditions to stimulate the cells, discard the liquid, take 100 μl and add it into PVDF 96-well plate, set 3 duplicate wells for each condition, and incubate for 16-20 hours at 37 ℃ and 5% CO2 incubator. 5) Add detection antibody: Discard the culture medium, wash with PBS containing 0.05% Tween20 for 4 times, and then 350 μl/well/time. (5) Addition of detection antibody: discard the culture medium, wash with PBS containing 0.05% Tween20 4 times, 350 μl/well/times, discard the liquid, dilute the detection antibody with the blocking solution, 100 μl/well, incubate at 4 ℃ overnight. 6) Color development: discard the liquid, wash with PBS containing 0.05% Tween20 4 times, 350 μl/well/times, discard the liquid, add the color developer, 100 μl/well, and develop the color for 10 minutes at room temperature. 7) Data reading: after the color development is completed, discard the color developer, wash 3 times/times with ddH2O, 2O, and add the color developer, 100 μl/well, and incubate at room temperature for 16-20 hours. 7) Read the data: After color development, discard the color solution, wash with ddH,2O for 3 times, dry the plate, and read the data with CLT plate reader. (4) Detection of immunoglobulin secreted by B cells by FACS method 1) Cell preparation: Isolate human PBMC or mouse spleen single nucleated cells, purify B cells. 2) Cell culture: Adjust the cell concentration to 2x10/mL with RPMI 1640 culture medium, select suitable culture conditions to stimulate the cells, take 200 μl and add it to 96-well round bottom culture plate, 3 wells for each condition, 37 ℃, 5% CO2, and then add 200 μl into 96-well round bottom culture plate, 3 wells for each condition, and then add 200 μl into 96-well round bottom culture plate. Incubate the cells at 37 ℃ in 5% CO2 incubator for 3-4 days. 3) Staining of smlg: After cell culture, wash the cells twice with staining buffer, and resuspend the cells with 100 μl of PBS/BSA containing 50 μg/mL anti-human/mouse FcYR IIb monoclonal antibody before labeling them with fluorescein-labeled anti-Ig antibody, incubate the cells at 4 ℃ for 15 minutes to remove non-specific staining; incubate the cells at 4 ℃ for 15 minutes with fluorescein-labeled anti-Ig antibody to remove non-specific staining; and incubate the cells at 4 ℃ for 15 minutes with fluorescein-labeled anti-Ig antibody. Incubate the cells with fluorescein-labeled anti-Ig antibody and other surface molecular antibodies (e.g. CD19, CD25, CD5, etc.) for 30 min at 4 ℃. 4) Intracellular staining of Ig: After cell culture, wash the cells twice with staining buffer, then wash the cells with PBS once, resuspend the cells with PBS at a concentration of 2x10/mL, and then add methanol/PBS to fix the cells (at a final concentration of 2%). The cells were fixed with methanol/PBS (final concentration of 2%) for 20 minutes at room temperature; the cells were washed once with staining buffer, resuspended with membrane-breaking solution, and the membrane was broken for 2 hours at 4 ℃/overnight; the cells were labeled with fluorescein-labeled anti-Ig antibody and other surface molecule antibodies (e.g., CD19, CD25, CD5, etc.), and the incubation was carried out at 4 ℃ for 30 minutes. 5) Flow cytometer detection: after incubation, the cells were washed once with 1 mL of staining buffer, and then washed with 200 μl of PBS/BSBS5. After incubation, cells were washed once with 1 mL of staining buffer, resuspended with 200 μl of PBS/BSA, and then detected and analyzed by flow cytometry.Caveat
(1) It is recommended to purify the initial B cells before stimulating them to exclude the influence of other cells on the stimulation.(2) The concentration of the stimulant LPS used in the experiment is related to the cell species. Generally, B cells from C57BL.10, BALB/c, or CBA strain mice and human B cells are responsive to LPS, and the concentration of the cells used in the stimulation should not be too high, and is generally controlled to be below 2x105/mL.
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