TITLE: T cell purification and activation
AUTHORS: Moustafa Nouh Elemeery, Salix Boulet, louis-eric.trudeau Trudeau, Nathalie Labrecque

[DESCRIPTION]
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 EasySep‱ magnet without the use of columns.

[STEPS]
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1. Dilute CD3 antibody (145-2C11) (clone KT3 can also be used) to 1 1621 in PBS.

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2. Coat plates with anti-CD3 antibody.

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3. 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. 

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4. Remove the antibody (aspirate) and wash 2 times with PBS.

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5. Add 100 µL of RPMIc if 96 wells or 1 mL if 24 wells and incubate the plate at 37 °Cfor 15 min . See complete recipe for RPMIc in Materials section.
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6. 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.
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3.1. Add 100 µL of antibody/well if 96 wells or 1 mL per well if 24 wells.

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3.2. Incubate the plate for 1440 min at 4 °C or 60 min at 37 °C.

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7. Purify CD8+ T cells using EasySep mouse naïve purification kit (STEMCELL, catalo # 19858) as follow:
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9. Resuspend cells in 5 mLof RPMIc, count cells and adjust the concentration to 2x106 cells/mL

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8. Wash cells with RPMIc medium.

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11. Add purified anti-CD28 antibodies to reach a concentration of 5 1621.

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10. Add 100 µL of cells (2x105 cells) per well if 96 wells or 1 mL of cells (2x106 cells) per well if 24 wells).

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12. Incubate the plate at 37 °C and 5% CO2 for 1440 min.

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7.1. Use a frosted microscope slide to homogenize spleens in PBS or Hanks' Balanced Salt Solution (HBSS) containing 2% fetal bovine serum (FBS).

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7.2. Remove aggregates and debris by passing cell suspension through a 70 μm mesh nylon strainer. Collect cells in a 15 mL tube.

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7.3. Centrifuge at 1300 rpm, 5 min and discard the supernatant.

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7.4. Red blood cells lysis is done by adding5 mL/spleen of 0.83% ammonium chloride and incubate for 5 min(or 2 min for ACK lysing buffer) at Room temperaturewhile continuously shaking tubes.

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7.5. Quench by filling up tube with RPMIc.

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7.6. Centrifuge at 1300 rpm, 5 minand discard supernatant.

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7.7. Resuspend in EasySep buffer at 1x108 cells/ml (Easysep mouse naïve CD8+ T cell isolation kit (#19858A) and follow the protocol provided by STEMCELL.
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13. Add 20 Unit/ml of IL-2.

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14. Incubate the plate at 37 °Cand 5% CO2 for another 2880 min.

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15.