T cell purification and activation
louis-eric.trudeau Trudeau, Moustafa Nouh Elemeery, Salix Boulet, Nathalie Labrecque
Disclaimer
ETHICS DISCLAIMER
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
This protocol details the purification and activation of Mouse Naïve CD8+ T Cell from mouse spleens using a purification kit. The cells are then activated by culturing them in plates coated with anti-CD3 antibodies and adding soluble anti-CD28 antibodies. The activation process occurs over 72 hours, with IL-2 added after the first 24 hours to support T cell proliferation and survival. The isolation kit from STEMCELL is designed to isolate naïve CD62L+CD44-CD8+ T cells from single-cell suspensions of splenocytes by negative selection. Unwanted cells are targeted for removal with biotinylated antibodies that are directed against non-naïve CD8+ T cells (CD4, CD11b, CD11c, CD19, CD44, CD45R/B220, CD49b, TCRγ/δ, TER119) and streptavidin-coated magnetic particles. Labeled cells are separated using an EasySepTM magnet without the use of columns.
Steps
Purification and activation
Dilute CD3 antibody (145-2C11) (clone KT3 can also be used) to 1 in PBS.
Coat plates with anti-CD3 antibody.
Use 24 well (Sarstedt, cat# 83.1836.500) or 96 well (Sarstedt, cat# 82.1581.001) plates. If these plates are not used, there is a risk of partial stimulation due to low absorbance of the antibody on the plate.
Add 100µL of antibody/well if 96 wells or 1mL per well if 24 wells.
Incubate the plate for 24h 0m 0s at 4°C or 1h 0m 0s at 37°C.
Remove the antibody (aspirate) and wash 2 times with PBS.
Add 100µL of RPMIc if 96 wells or 1mL if 24 wells and incubate the plate at 37°Cfor 0h 15m 0s . See complete recipe for RPMIc in Materials section.
Collect spleen from mice 6-8 weeks in complete RPMI media (RPMIc), sex matched with recipient mice.
Note: using spleens from 6-8 week old mice ensures that the isolated T cells are from young adult mice with a fully developed immune system, and the sex-matching helps prevent potential complications in downstream applications or experiments.
Purify CD8+ T cells using EasySep mouse naïve purification kit (STEMCELL, catalo # 19858) as follow:
Use a frosted microscope slide to homogenize spleens in PBS or Hanks' Balanced Salt Solution (HBSS) containing 2% fetal bovine serum (FBS).
Remove aggregates and debris by passing cell suspension through a 70 μm mesh nylon strainer. Collect cells in a 15 mL tube.
Centrifuge at 1300rpm and discard the supernatant.
Red blood cells lysis is done by adding5mL/spleen of 0.83% ammonium chloride and incubate for 0h 5m 0s(or 0h 2m 0s for ACK lysing buffer) at Room temperaturewhile continuously shaking tubes.
Quench by filling up tube with RPMIc.
Centrifuge at 1300rpmand discard supernatant.
Resuspend in EasySep buffer at 1x108 cells/ml (Easysep mouse naïve CD8+ T cell isolation kit (#19858A) and follow the protocol provided by STEMCELL.
Wash cells with RPMIc medium.
Resuspend cells in 5mLof RPMIc, count cells and adjust the concentration to 2x106 cells/mL
Add 100µL of cells (2x105 cells) per well if 96 wells or 1mL of cells (2x106 cells) per well if 24 wells).
Add purified anti-CD28 antibodies to reach a concentration of 5.
Incubate the plate at 37°C and 5% CO2 for 24h 0m 0s.
Add 20 Unit/ml of IL-2.
Incubate the plate at 37°Cand 5% CO2 for another 48h 0m 0s.
