T lymphocyte proliferation assay

Summary

This module introduces the mouse T lymphocyte proliferation assay.

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

Operation method

T lymphocyte proliferation assay

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Basic scenario 1 Activation of un-sensitized T lymphocytes Material

Option 1 Activation of unsensitized T cells with antibodies

This approach is used when the anti-CD3/TCR complex antibody does not bind effectively to the Fc receptor on mouse helper cells or when its soluble form does not effectively activate T cells. It should be noted that in cases where a particular antibody (e.g., anti-G 7, Thy-I monoclonal antibody) has difficulty in inducing a proliferative response in T cells, Basic Protocol 1 is recommended.

Additional material

P B S (room temperature and 4°C)

Prepare lm g/m l of purified anti-CD3 or anti-TCR monoclonal antibody (non-specific activation of T cells) or lm g/m l of purified anti-V news or anti-TCR-yS monoclonal antibody (specific activation of T cells, Table 1) with PBS.
2.11.1)

1. In a 4 ml polystyrene conical-bottom centrifuge tube, use sterile room-temperature PBS (protein-free) to mix lmg/ml of sterile monoclonal antibody storage solution (Table 2.11.1) with 4 concentrations of the working solution according to the experimental needs: e.g. lOOug/ml, 10ug/ml, 1ug/ml, O.lug/ml, and make it up before use.

2 . Add 30 ul of each concentration of Antibody Working Solution (up to 2-3 ug per well; optimal concentration is usually 10ug/ml) to a 96-well round bottom plate (one row for each monoclonal antibody) and use 30/xl of PBS as a control well.

3 . Cover the plate and tap the side of the plate to ensure that the bottom of the wells are completely covered with liquid. Incubate at 37°C for 90 min or 4°C overnight. Proceed with the next step while incubating.

4 . Prepare a reaction cell suspension (highly purified, but the auxiliary cells do not affect the results of the experiment) as in Steps 1 to 3 of Basic Protocol 1.

5 . Add 200ul of pre-cooled PBS to each well and wash the wells of the above antibody-coated culture plate, invert the plate in the ultra-clean bench and tap it on absorbent paper to remove the residual liquid, and re-wash the plate 2 or 3 times to ensure that the excess antibody is removed.

6 . Add 2 X ¹⁰⁵ cells/(0.2 ml-well) of the prepared cell suspension to the washed plate (to be determined empirically; hybridoma supernatant should not be used). If the cells are not ready at the moment, add 100M PBS and store at 4°C overnight (PBS should be removed before adding cells).

7 . Follow steps 7 to 9 in the basic protocol1 , and add ^3H TdR after 2 to 3d of incubation (perform kinetic experiments to determine the optimal time).

Option 2 Mixed lymphocyte proliferation assay Additional material
Adjuvant regimen 1 Elimination of T lymphocytes from antigen-presenting or stimulating cell suspensions

The main purpose of this method is to exclude helper T cells. Methods for purification and enrichment of antigen-presenting cells are described in the relevant sections; the methods described in Module 2.3 do not exclude T cells and are therefore not recommended.

Additional materials (see Basic Scheme 1 for additional materials)

Splenocytes from unimmunized mice genetically homologous to the reactive T cells

H B S S

Low-toxicity rabbit supplement (Cedarlane), diluted in ice-cold sterile deionized water

Anti-Thy-l.2 monoclonal antibody (H ○ -13-4, ATCCNO . TIB9 9 ) or anti-Thy-l.l monoclonal antibody (H ○ -22-l , A T C C n o . TIB1 0 0 ; or other anti-Thy-I monoclonal antibodies See C P I M Table 3.4.1 and Ascitic fluid preparation.

Adjuvant regimen 2 Inactivation of adjuvant/stimulated cells

Note: Most investigators preferentially use mitomycin C to inactivate cells when antigenic differences in the genetic code need to be examined or when an irradiated source is not available.

Mitomycin C Inactivated Cells

Additional Material

Cells maintain antigen-presenting function; after 1100 ~ 2000 rad radiation, antigen-presenting ability decreased dramatically; > 20,000 m d inactivation of B cells lost APC function. On the other hand, macrophages and dendritic cells still maintain the function of antigen presentation after 3000 rad irradiation. To ensure that B cells do not participate in the response, some researchers prefer 2000 rad. However, when detecting antigens that stimulate the cellular MLS locus, < 1000 m d of radiation should be used because B cells are more efficient at presenting MLS antigens. Alternatively, the Mls response can be detected after mitomycin C treatment, which maintains the antigen-presenting function of the treated B cells.

When transformed cell lines are used as antigen presenting or helper cells, high intensity irradiation is used to block their proliferation. The appropriate radiation intensity for each cell line needs to be determined on a pre-test basis, but should be at least 5000 rad; some transformed cell lines may require up to 10,000 to 20,OOO rad, or some may be more sensitive to mitomycin C. The radiation intensity for each cell line should be determined on a pre-test basis, but should be at least 5000 rad; some transformed cell lines may require up to 10,000 to 20,OOO rad or some may be more sensitive to mitomycin C.

Basic Scheme 2 Activation of sensitized T cells Material

(It reaches its peak on day 4 or 5).

Basic protocol 3: Detection of activation and inhibition of C D 4+C D 25+ T cells. Material
Option 3 C D 4+C D 25+ A two-step assay of T cell suppressive function: short-term activation and expansion

C D 4+C D 25+ T cells and analyze their suppressive function

Additional Material

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