T cell differentiation from mice spleen tissue
Ningbo Zheng, Weiyi Peng
Abstract
This protocol is for T cell differentiation from mice spleen tissue to investigate T cell function (including differentiation and proliferation) in vitro.
Steps
Day 0
Prepare precoated 96-well plate using antibodies described in materials section.
Day 1
Prepare single-cell suspensions of mice spleen tissues as per steps below
Euthanize mice by CO2 inhalation or other means of euthanasia.
Collect the spleen from the mice.
Put a 40μm vacuum filter on a 50mL tube. Mesh the spleen on the strainer using the back of a syringe.
Add 5mL of RPMI medium to the mashed spleen to collect splenocytes into a 50mL tube.
Spin down cells at 1500rpm for 3min. Discard supernatant.
Add 0.5mL ACK lysis buffer to the red cell pellet, resuspend, and wait 30 seconds to lyse RBC.
Directly add 5mL T cell medium.
Spin down cells at 1500rpm for 3min. Discard supernatant.
Prepare a new 50mL tube with a new 40μm vacuum filter.
Resuspend cells in 10mL T cell medium and filter through a new 40μm vacuum filter.
Count the cells.
Isolation of naïve CD4+ T cells from single-cell suspensions from spleen tissue on Day 1
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).
T cell differentiation on Day 1
Seed the cells into plate and culture with different conditions
Regulate the cell concentration at 5×105/mL with T cell medium
| A | B | C |
|---|---|---|
| Naïve CD4+ T cells | 5×104/well | 100μL/well |
| T cell medium | 50μL/well | |
| Total | Total | 150μL/well |
Take out the pre-coated plate from 4℃.
Remove the supernatant and add 200μL PBS to the pre-coated plate and then remove PBS to wash the coated plate. Repeat 2 times.
Seed wells of a 96-well plate with 150μL cells cocktail (100μL cells and 50μL T cell media.
Seed 100μL differentiation cocktail.
Incubate for 5 days at 37°C.
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)
Collect cells and count.
Regulate cell concentration at 1×106/mL.
Seed 100μL cells into a new 96-well plate (U bottle)
Add 100μL PMA cocktail and culture overnight. For non-stimulate cells, add 100μL T cell medium.
| A | B | C |
|---|---|---|
| Final concentration | Amount | |
| T cells | 1×106/mL | 100μL |
| T cell Medium | n.a | 100μL |
| PMA (50μg/mL) | 50ng/mL | 0.2μL |
Collect supernatant for ELISA. (Note: The supernatant can be frozen at -20°C.)
Detect Treg cell differentiation by Flow cytometry on Day 5
Spin down cells at 1500rpm for 3min at RT.
Remove the supernatant over the sink in one motion.
Add 150μL FACS buffer and spin down cells at 1500rpm for 3min at RT. Remove the supernatant.
Surface staining, add 50μL FACS Buffer mAbs cocktail:
| A | B |
|---|---|
| FACS buffer | 50µL |
| Anti-CD4-eFluor 450 | 0.3µL |
| Anti-CD25-PerCP | 0.3µL |
Incubate at 4°C for 30min in the dark.
Directly add 150uL FACS buffer and spin down cells at 1500rpm for 3min at RT.
Remove the staining buffer and thoroughly resuspend cells.
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).
Centrifuge samples at 400-600g for 5min at RT.
Add 200µL 1×Permeabilization Buffer (make fresh) to each well and centrifuge samples at 400-600g for 5min at RT. Discard the supernatant.
Add 50uL 1×Permeabilization Buffer mAb cocktail (Foxp3):
| A | B |
|---|---|
| 1×Permeabilization Buffer | 50µL |
| Anti-Foxp3-PE | 0.3µL |
Incubate at 4°C for 30min in the dark.
Directly add 200µL 1×Permeabilization Buffer to each well and centrifuge samples at 400-600g for 5min at RT. Discard the supernatant.
Add 200μL 1×Permeabilization Buffer for Flow Cytometry.
