Isolation and purification of T cell subsets

Summary

Isolated T cells are categorized into CD4+ and CD8+ T cells using anti-CD8 antibody. Since both types of cells can be

recovered, this method is superior to antibody and complement lysis methods

Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from "Comprehensive Immunology Laboratory Guide".

Operation method

Isolation and purification of T cell subsets

Move

Basic Programs Indirect Amalgamation Separation of T Cell Subpopulations Materials

Affinity-purified human immunoglobulin-adsorbed sheep anti-mouse Ig (Tago)

0.05m ol/L Tris -Cl, pH 9. 5

Anti-CD 8 antibody or other specific mouse antibody (polyclonal or monoclonal; e.g., Coulter or Becton Dickinson)

PBS

Total T cells (see supplemental protocols 1 or 2)

PBS with 5 % and 1 % (VAO heat-inactivated FCS (56°C, Ih))

R PM I complete medium (serum-free, filtered to maintain sterility)

15 mmX IOOmm plastic petri dishes, bacteriological grade (not for tissue culture)

15 ml centrifuge tubes

Beckman GPR GH-3. 7 Flat Head Centrifuge (or equivalent), 4 to 10°C

Benchtop Shaker, 4°C

Inverted microscope

sterile cell scraping

1. Dilute sheep anti-mouse Ig with 0-05m ol/L Tris-Cl of pH9.5 to 10 ug/ml, take IOml and add it to 15 mmXIOOmm Petri dish, gently shake it to cover the bottom of the dish, incubate at room temperature for 40 min, or incubate at 4°C for 24 h. Incubate for 10 min at room temperature, or incubate for 24 h at 4°C for 10 min.

Option: The antibody relies on complement-mediated cytotoxicity to isolate T cell subsets.

A subpopulation of T cells can be removed by binding to a specific antibody, in this case the anti-CD4 antibody.

Additional Material

Newborn rabbit supplement (Cedarlane Laboratories), no repeated freezing and thawing allowed

R P M I-I Complete Medium

Anti-C D 4 antibody or other specific mouse antibody (polyclonal or monoclonal; must be complement-binding IgG2a or I g M ; e.g., Coulter or Becton Dickinson)

1. Detect nonspecific toxicity of neonatal rabbit complement to human mononuclear cells. Determine the concentration of complement (usually 20 % to 50 %) required to produce maximal antibody-mediated cytotoxicity and minimal nonspecific cytotoxicity (e.g., cytolysis in the absence of specific antibody).

2. Determine the amount of antibody required to label individual nucleated cells for flow cytometry.

3. Add ^IO6~IO7 T cells into centrifuge tube and centrifuge at 400 g for lOmin at 18~20°C. Discard the supernatant and resuspend the cells.

Option 1: Isolation of T cells by rosette formation from neuraminidase-treated sheep erythrocytes. Materials

Sheep erythrocytes suspended in Alsever's solution (S R B C ; Colorado Serum).

H B S S

l U/ml neurotransaminase (low-pressure lyophilized powder, type X; Sigma) dissolved in PBS (Appendix I); 1 ml dispensed and stored at 20°C.

RPMI-10 complete medium

P B M C dissolved in R P M I -10 complete medium (I X ^IO7 cells/m l; Unit 8.1)

Fetal bovine serum (FCS; heat inactivated at 56°C for Ih)

Polysucrose-pantethine glucosamine solution

AC K lysis solution (optional)

Beckman G P R G H -3.7 Flat-head centrifuge (or other similar equipment)

15 ml and 50 ml conical-bottom tubes (e.g. Falcon)

15 ml round bottom centrifuge tubes (e.g. Falcon)

1. Add 15-25 ml of sheep red cells (SRBC) suspended in Alsever solution to a 50 ml centrifuge tube, then fill with HBSS and centrifuge at IOOOg for 10 min at 18-20°C. Discard the supernatant, resuspend the cells with HBSS, and centrifuge again.
Centrifuge again and repeat the process. Store the washed SRBC for 2~3d.

2 . Add Iml S RBC suspension to a 50 ml centrifuge tube and add Iml l U/mL neuraminidase dissolved in PBS, pipette to homogenize the cells and incubate at 37°C for 1h.

3. Fill with HBS, centrifuge as above, discard the supernatant, and rewash twice. Add 49 ml of RPMI-10 complete medium to resuspend the cells (final concentration of SRBC is 2%, V/V). Store at 4°C until use (5-7d).

4. Add <≤2 X ^107PBMC to a 15 ml round-bottomed centrifuge tube and centrifuge at 400 g for 10 min at 18-20°C. Discard the supernatant and resuspend the cells in RPMI-10 to a concentration of IX ^IO7 cells/ml.

5 . Incubate with 2 ml of heat-inactivated FCS and 2 ml of neuraminidase-treated SR-BC per IX ^IO7 cells/ml of PBMC in a water bath at 37°C for 1 h. Incubate at 4°C for 5 min at 200 g. Incubate on ice for Ih (no need to discard supernatant).

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