Human T cell activation

Human PBMCs can be activated in vitro by soluble anti-human CD3 antibodies. We have performed titration studies with these antibodies and established the following protocol for stimulation of PBMC.

Materials

1X sterile PBS

Anti-human CD3:

Clone OKT3 (Functional Grade Cat. No. 16-0037) or Clone HIT3a (Functional Grade Cat. No. 16-0039)

Complete RPMI-1640

Sterile PBMC

96-well flat-bottom microtiter plates with lids (Costar Cat. No. 3596)

MTT Buffer

MTT Lysing Solution

Instruments

Pipettes and pipettors, Multichannel pipettor

Centrifuge

37°C, CO2 incubator

96-well micro test spectrophotometer

Experiment Duration

30 minutes preparation of PBMC

20 minutes to set up the assay

2-4 days incubation

Method

Prepare PBMC and resuspend the cells at 1-2x106/ml of complete RPMI. Note: This density of PBMC gives a good response. If experimental conditions require, a titration of cell densities (2-3x106/ml to 105/ml) should be performed for optimization.

Add 100l of the cell suspension to each well. For each condition, use triplicate wells.

Add soluble antibody in 100l to each well. Titrate antibodies for optimal performance in the assay conditions used. If isolated T cells are to be used in proliferation assays, we recommend using these antibodies at 10g/ml immobilized on plastic (i.e. bound to wells of 96-well assay plates).

Place in a humidified 37°C, 5% CO2 incubator.

Incubate for 2-4 days. Note: Proliferation of cells between day 2 and 4 gives a good response; however, this incubation time can also be optimized for specific experimental conditions.

Add 20l of the MTT buffer to each well and put back in the incubator for 4 hours.

Add 50l of the MTT Lysis Solution to each well, vortex gently and incubate overnight.

Read the plate at 570nm the next day.

Calculate the mean and standard errors and graph the data.

Buffers & Media

Complete RPMI-1640:

900ml RPMI-1640 (Hyclone Cat. No. SH30027.02)

100ml FBS (Hyclone Cat. No. SH30151.03) Heat inactivated (10% final)

1ml 2-mercaptoethanol (Gibco BRL Cat. No. 21985-023)

10ml L-Glutamine (Hyclone Cat. No. SH30034.01)

Antibiotic cocktail (optional)

MTT Buffer:

MTT (Sigma Cat. No. M5655) 5mg/ml stock solution in 1X PBS - Keep protected from light.

MTT Lysing Solution:

20% SDS 50% DMF