Presentation of exogenous antigens to T cells
Presentation of exogenous antigens to T cells
For granular antigens or liposome-encapsulated antigens, macrophages were applied as antigen-presenting cells. Author: J.E. Colligan et al, Translator: Xuitao Cao et al. This experiment is from the "Compendium of Immunology Laboratory Guide".
Operation method
Transmission of exogenous antigens to T cells Move Basic Program A test system for antigen processing and presentation Materials Proliferative (e.g., transformed B cells) or nonproliferative antigen-presenting cells that match the MHC of T hybridoma cells (Unit 7.1) D M E M -1 0 Complete Medium, 37°C T hybridoma cells in logarithmic growth phase Antigen: water-soluble protein or peptide (for water-insoluble peptides, dissolution in DMSO and solvent control are required) IL-2 E L ISA kit (e.g. Genzyme) or see Module 5.1 9 6-well flat-bottomed culture plates Sorvall RT- 6 0 0 0 Refrigerated Tabletop Centrifuge with HB-IOOO Rotor 0.22um low protein binding membrane for peptide filtration only Multi-channel pipettes and sterile tips la. For non-proliferating antigen-presenting cells: Add isolated celiac cells or other non-proliferating or low-proliferating cells to a 96-well flat-bottomed culture plate, 2 X IO5 cells/well, and adjust the volume of liquid to lOOul/well with DMEM-IO Complete Medium. Ib. For proliferative cell lines: Prepare B-cell suspensions (5 X IO 5 cells/ml) in DMEM-10 Complete Medium and add cells to 96-well flat-bottomed plates at 100ul/well (5 X IO 4 cells/ml). 2 . Collect T hybridoma cells in logarithmic growth phase (5 X 105 to IO6 cells/ml) by centrifugation at 400 to 500 g at room temperature, suspend the cells in pre-warmed DMEM-10 complete medium, count the cells (Appendix 3A), and adjust the concentration of the cells to 2 X 106 cells/ml. Add 50 ul/well ( IO5 cells/well) to a 96-well flat-bottom plate containing antigen-presenting cells and incubate at 37°C or proceed immediately to step 3. 3 . Prepare lmmol/L protein or peptide storage solution. Dissolve the synthesized peptides in deionized water and filter through a 0.22um low protein-binding membrane (can be stored at 4°C for several months). For protein antigens, they can be dissolved in DMEM-10 complete medium prior to use. 4 . Serial gradient dilutions of antigens in DMEM-10 complete medium can be designed according to the concentration required, with the antigen concentration being more than four times the final concentration. 5. Add 50ul/well of antigen to a 96-well flat-bottom plate containing antigen-presenting cells and T hybridoma cells. Three replicate wells are set up for each antigen concentration, as well as a series of blank control wells (filled with 50ul/well of DMEM-10 complete medium). Continue to incubate at 37C for 20-24 hours. Antigen, antigen presenting cells and T cells can be added in any order. 6. Centrifuge the plate at 400 to 500 grams per well and transfer 100 ul/well of supernatant to a new round-bottomed 96-well plate using a multichannel pipette. Store the original plate at 20°C for backup in case of problems with the IL-2 assay. 7. Freeze at -80°C for I h or at 1 20°C overnight to inactivate viable cells mixed into the supernatant (plates can be frozen at 1 20°C for several weeks). Detect the level of IL-2 in the supernatant (Box 5.1). This protocol can be used to detect antigenic peptides presented to T-hybridoma cells by MHC class I or MHC class II molecular pathways. DMEM culture medium (serum-free) 2 % (m A O paraformaldehyde solution dissolved in P B S Lysine wash la. For adherent peritoneal macrophages: add isolated peritoneal macrophages at 2 X 105 cells/well to 96 wells. lb. Other wall-adherent antigen-presenting cells: Add cells to 96-well flat-bottomed culture plates at 2 X 105 cells/well and adjust the total amount of liquid to 100ul/well with DMEM-10 Complete Medium. Incubate at 37°C for sufficient time to allow the cells to adhere to the wall. For cell lines that require overnight incubation for wall attachment, the cell concentration should be 2 to 4 times lower to allow for cell proliferation. 2 . Blow up the suspended cells with a multichannel pipette, aspirate the supernatant from one or more rows of culture plate wells at a time and immediately add 1-free D M E M 100ul/well. The remaining cells were treated in the same way. 3 . Dilute 2 % paraformaldehyde solution with serum-free DME (1 % paraformaldehyde) and proceed immediately to step 4. If solid solution toxicity is a problem in a subsequent step, dilute paraformaldehyde in DMEM at 0.5 %. 4. Replace the cell culture medium in the plate with fixative (IOOul/well). Incubate at room temperature for 1 〇 ~15 min. 5 . Dispense the fixative with a multichannel pipette and add 120ul/well of DMEM. To prevent the fixative from remaining, the pipette tip should avoid contacting the wall of the wells and avoid spilling. Discard DMEM and add lysine wash (120ul/well). Incubate for 20-30 min at room temperature. 6 . Wash the plates 4 times with DMEM (the amount of DMEM should be increased gradually, e.g. 150 Ul/well for the first two washes and 180 ul/well for the last two washes) to remove paraformaldehyde. After washing, add IOOiUl DMEM-10 complete medium to each well. 7 . Co-culture the fixed cells with hybridoma cells and antigens (see basic protocol, steps 2 to 5). After overnight incubation, check the viability of the T cells in the culture plate (residual fixative may cause T cell death). 8 . Collect the supernatant and assay the IL-2 level (see steps 6 to 7 of the basic protocol). Transformed B cells or other non-adherent cells are used as antigen-presenting cells. D M E M culture base (serum-free) 2 % (m/V ) paraformaldehyde solution, dissolved in I3BS Lysine Wash 15m l and 50m l sterile centrifuge tubes 1 . Collect mouse B lymphoma cells in the logarithmic growth phase, 400 to 500 cells, at room temperature, and centrifuge for lOmin. Suspend the cells in serum-free DMEM (5 X IO6 cells/ml). 2. Add equal amount of 2% paraformaldehyde solution, incubate at room temperature for 5~lOmin, 400~500 g at room temperature, centrifuge for lOmin, discard supernatant and suspend cells in DMEM (the amount of liquid should be greater than or equal to that of the fixative), then incubate at 400~500 g at room temperature, centrifuge for lOmin. 3 . Suspend the cells in lysine solution and incubate at room temperature for 20~30 min. After centrifugation and discarding the supernatant, wash the cells with DMEM for two times. 4 . Suspend the cells in DMEM complete medium (2 X IO6 cells/ml) and add the cells into 96-well flat-bottomed culture plates (2 X 105 cells/well). 5 . Co-culture the fixed cells with the hybridoma cells and antigen (see basic protocol, steps 2 to 5). After overnight incubation, check the viability of the T-cells in the culture plate (residual fixative will cause some T-cells to die). 6 . Collect the supernatant and assay the IL-2 level (see basic protocol, steps 6 to 7). 2 % (m/V) paraformaldehyde solution, dissolved in PBS Lysine Wash 1. Add antigen-presenting cells (e.g., isolated peritoneal macrophages) to 96-well flat-bottomed plates at 2 X 105 cells/well and incubate to make them adherent to the wall (e.g., macrophages should be incubated at 37°C for 2 hours). After blowing the supernatant, the supernatant was replaced with 2 . Dilute the antigen in a continuous gradient with DMEM-10 Complete Medium at a concentration greater than two times the working concentration. Add the diluted antigen to a plate (100ul/well) lined with antigen-presenting cells, including two rows of antigen-free control wells (see step 7). Incubate at 37°C for a period of time (e.g. 2 h for activated peritoneal macrophages). 3 . At the end of the incubation, mix equal parts of serum-free DMEM and 2% paraformaldehyde solution (1 % paraformaldehyde concentration) and proceed immediately to the next step. If toxicity of the fixative is a problem in subsequent operations, consider using a lower concentration of paraformaldehyde solution (e.g. 0.5 %). 5 %) 4 . Aspirate the culture supernatant, wash the cells once with DMEM (200^1/well), add fixing solution (100ul/well) and fix for 10~15 min at room temperature. 5 . Dispose of the fixative with a multichannel pipette and add 120m 1/well of DME, avoiding the tip of the pipette from touching the wall of the wells and avoiding spilling of the fixative in order to prevent the fixative from being left behind. Dispose of DMEM and add lysine wash (120ul/well). Incubate for 20~3Qmin at room temperature. 6 . Wash the plate 4 times with DMEM (the amount of DMEM used should be gradually increased, e.g. 150ul/well for the first two washes and 180u1/well for the last two washes) to remove the residual paraformaldehyde. After washing, add IOOmI DMEM-10 complete medium to each well. 7 . Suspend T cell hybridoma cells in D M E M complete medium ( IO6 cells/ml). Add to cell culture plate ( 105 cells/well). In a row of control wells (without antigen), add 20ul/well of immunogenic peptide as positive test wells, and the final concentration should be 10 times of the working concentration (the total volume of the culture wells is 220ul at this time, but it does not affect the results of the experiment). Incubate at 37°C for 20~24 h. 8 . Collect the supernatant for IL-2 level (see basic protocol, steps 6 to 7). For more product details, please visit Aladdin Scientific website.
For wall-adherent peritoneal macrophages: Isolate peritoneal macrophages at 2 X 105 cells/well into 96-well flat-bottomed culture plates and adjust the total amount of liquid to 100 ul/well with DMEMI-IO Complete Medium. The cells were incubated at 37°C for 2 hours to allow the cells to adhere to the wall.
After blowing the supernatant, replace the supernatant with DMEM-IO complete medium (IOOm I/well).