TITLE: T cell differentiation from mice spleen tissue
AUTHORS: Ningbo Zheng, Weiyi Peng

[DESCRIPTION]
This protocol is for T cell differentiation from mice spleen tissue to investigate T cell function (including differentiation and proliferation) in vitro.

[STEPS]
SECTION: Day 0
1. Prepare precoated 96-well plate using antibodies described in materials section.
SECTION: Day 1
2. Prepare single-cell suspensions of mice spleen tissues as per steps below
SECTION: Day 1
3. Euthanize mice by CO2 inhalation or other means of euthanasia.
SECTION: Day 1
4. Collect the spleen from the mice.
SECTION: Day 1
6. Add 5mL of RPMI medium to the mashed spleen to collect splenocytes into a 50mL tube. 
SECTION: Day 1
5. Put a 40μm vacuum filter on a 50mL tube. Mesh the spleen on the strainer using the back of a syringe. 
SECTION: Day 1
10. Spin down cells at 1500rpm for 3min. Discard supernatant.
SECTION: Day 1
9. Directly add 5mL T cell medium.
SECTION: Day 1
8. Add 0.5mL ACK lysis buffer to the red cell pellet, resuspend, and wait 30 seconds to lyse RBC.
SECTION: Day 1
7. Spin down cells at 1500rpm for 3min. Discard supernatant.
SECTION: Day 1
13. Count the cells.
SECTION: Day 1
12. Resuspend cells in 10mL T cell medium and filter through a new 40μm vacuum filter.
SECTION: Day 1
11. Prepare a new 50mL tube with a new 40μm vacuum filter.
SECTION: Isolation of naïve CD4+ T cells from single-cell suspensions from spleen tissue on Day 1
14. Naïve CD4+ T cells were isolated from single-cell suspensions from spleen tissue by negative selection using the EasySep Mouse CD4+ T-cell Isolation Kit (#19852, STEMCELL Technologies, Vancouver, Canada).
SECTION: T cell differentiation on Day 1
15. Seed the cells into plate and culture with different conditions
SECTION: T cell differentiation on Day 1
17. Take out the pre-coated plate from 4℃.
SECTION: T cell differentiation on Day 1
16. Regulate the cell concentration at 5×105/mL with T cell medium
 

SECTION: T cell differentiation on Day 1
21. Incubate for 5 days at 37°C.
SECTION: T cell differentiation on Day 1
20. Seed 100μL differentiation cocktail.
SECTION: T cell differentiation on Day 1
19. Seed wells of a 96-well plate with 150μL cells cocktail (100μL cells and 50μL T cell media.
SECTION: T cell differentiation on Day 1
18. Remove the supernatant and add 200μL PBS to the pre-coated plate and then remove PBS to wash the coated plate. Repeat 2 times.
SECTION: Detect T cell differentiation by ELISA on Day 5 and Day 6 (Note: The chosen time point is not fixed, according to the experimental purpose)
22. Collect cells and count.
SECTION: Detect T cell differentiation by ELISA on Day 5 and Day 6 (Note: The chosen time point is not fixed, according to the experimental purpose)
26. Collect supernatant for ELISA. (Note: The supernatant can be frozen at -20°C.)
SECTION: Detect T cell differentiation by ELISA on Day 5 and Day 6 (Note: The chosen time point is not fixed, according to the experimental purpose)
25. Add 100μL PMA cocktail and culture overnight. For non-stimulate cells, add 100μL T cell medium.
 
 
SECTION: Detect T cell differentiation by ELISA on Day 5 and Day 6 (Note: The chosen time point is not fixed, according to the experimental purpose)
24. Seed 100μL cells into a new 96-well plate (U bottle) 
SECTION: Detect T cell differentiation by ELISA on Day 5 and Day 6 (Note: The chosen time point is not fixed, according to the experimental purpose)
23. Regulate cell concentration at 1×106/mL.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
27. Spin down cells at 1500rpm for 3min at RT.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
30. Surface staining, add 50μL FACS Buffer mAbs cocktail:
 
 
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
29. Add 150μL FACS buffer and spin down cells at 1500rpm for 3min at RT. Remove the supernatant.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
28. Remove the supernatant over the sink in one motion.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
37. Add 50uL 1×Permeabilization Buffer mAb cocktail (Foxp3):
 
 
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
36.  Add 200µL 1×Permeabilization Buffer (make fresh) to each well and centrifuge samples at 400-600g for 5min at RT. Discard the supernatant. 
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
35. Centrifuge samples at 400-600g for 5min at RT.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
34. Add 200µL of Foxp3 Fixation/Permeabilization working solution to each well for 1 hour at RT in the dark. (4×stock solution must be diluted prior to use with the Fixation/Permeabilization Diluent dilute 1 part concentrate with 3 parts diluents, make fresh).
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
31. Incubate at 4°C for 30min in the dark.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
33. Remove the staining buffer and thoroughly resuspend cells.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
32. Directly add 150uL FACS buffer and spin down cells at 1500rpm for 3min at RT.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
40. Add 200μL 1×Permeabilization Buffer for Flow Cytometry.
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
39. Directly add 200µL 1×Permeabilization Buffer to each well and centrifuge samples at 400-600g for 5min at RT. Discard the supernatant. 
SECTION: Detect Treg cell differentiation by Flow cytometry on Day 5
38. Incubate at 4°C for 30min in the dark.