Feb 29, 2024
T cell differentiation from mice spleen tissue
- Ningbo Zheng1,2,
- Weiyi Peng1,2
- 1Department of Biology and Biochemistry, University of Houston, Houston, TX, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network
Protocol Citation: Ningbo Zheng, Weiyi Peng 2024. T cell differentiation from mice spleen tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.261gedy8ov47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 29, 2024
Last Modified: February 29, 2024
Protocol Integer ID: 95978
Keywords: mice spleen tissue, spleen tissue, including differentiation, tissue
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: Grant ID: ASAP-000312
Abstract
This protocol is for T cell differentiation from mice spleen tissue to investigate T cell function (including differentiation and proliferation) in vitro.
Materials
1. Pre coating of 96 well plate with antibodies using following reagents
| A | B | C | |
| Reagent | Final concentration | Amount | |
| PBS | n/a | 100mL/well | |
| Anti-CD3e (0.5mg/mL) | 2.5mg/mL | 0.5mL/well | |
| Anti-CD28 (1mg/mL) | 1mg/mL | 0.1mL/well |
2. Prepare FACS buffer and T cell culture media (See Appendix 1 below)
| A | B | C | |
| FACS buffer | |||
| Reagent | Final concentration | Amount | |
| PBS | n/a | 490mL | |
| FBS | 2% | 10mL | |
| Total | n/a | 500mL |
Note: Can be prepared in advance and stored at 4℃ for at least 4 months. Filtration through a
0.2 μm vacuum filter is recommended.
| A | B | C | |
| T cell culture media | |||
| Reagent | Final concentration | Final concentration | |
| MEM⍺ | n/a | 429mL | |
| FBS | 10% | 50mL | |
| GlutaMAX 100× | 2× | 10mL | |
| HEPES, 1M | 0.02M | 10mL | |
| 2-mercaptoethanol (55 mM) | 55μM | 0.5mL | |
| Normocin, 1g (50mg/ml) | 50μg /ml | 0.5mL | |
| Total | n/a | 500mL |
Note: Can be prepared in advance and stored
at 4℃ for up to 1 month.
2. Prepare T-cell differentiation cocktails [1-4] (Appendixes 2 below)
| A | B | C | |
| Th0 differentiation cocktail (2.5×) | |||
| Reagent | Final concentration | Amount | |
| T cell culture media | n/a | 1mL | |
| IL-2 (5×105U/mL) | 100U/mL | 0.5mL |
| A | B | C | |
| Th1 differentiation cocktail (2.5×) | |||
| Reagent | Final concentration | Amount | |
| T cell culture media | n/a | 1mL | |
| IL-2 (5×105U/mL) | 100U/mL | 0.5mL | |
| IL-12 (10μg/mL) | 4ng/mL | 1mL | |
| Anti-IL-4 (10mg/mL) | 10μg/mL | 2.5mL |
| A | B | C | |
| Th2 differentiation cocktail (2.5×) | |||
| Reagent | Final concentration | Amount | |
| T cell culture media | n/a | 1mL | |
| IL-2 (5×105U/mL) | 100U/mL | 0.5mL | |
| IL-4 (10μg/mL) | 10ng/mL | 2.5mL | |
| Anti-IFNg (10mg/mL) | 10μg/mL | 2.5mL |
| A | B | C | |
| Th9 differentiation cocktail (2.5×) | |||
| Reagent | Final concentration | Amount | |
| T cell culture media | n/a | 1mL | |
| IL-2 (5×105U/mL) | 100U/mL | 0.5mL | |
| IL-4 (10μg/mL) | 10ng/mL | 2.5mL | |
| Anti-IFNg (10mg/mL) | 10μg/mL | 2.5mL | |
| TGF-b (5μg/mL) | 1ng/mL | 0.5mL | |
| IL-1b (10μg/mL) | 10ng/mL | 2.5mL |
| A | B | C | |
| Treg differentiation cocktail (2.5×) | |||
| Reagent | Final concentration | Amount | |
| T cell culture media | n/a | 1mL | |
| IL-2 (5×105U/mL) | 300U/mL | 1.5mL | |
| Anti-IFNg (10mg/mL) | 10μg/mL | 2.5mL | |
| TGF-b (5μg/mL) | 5ng/mL | 2.5mL | |
| Anti-IL-4 (10mg/mL) | 10μg/mL | 2.5mL |
Note: Cocktails can be prepared in advance and stored at 4℃ for several days, seed 100mL in each well.
3. Reagents or Resources
| A | B | C | |
| MEM⍺ | Fisher Scientific | Cat#32-571-101 | |
| FBS | R&D Systems | Cat#S11150 | |
| GlutaMAX 100× | Fisher Scientific | Cat#35050061 | |
| HEPES | Fisher Scientific | Cat#MT25060CI | |
| 2-mercaptoethanol | Fisher Scientific | Cat#21985023 | |
| Anti-CD3e | Cytek Biosciences | Cat#145-2C11 | |
| Anti-CD28 | BD Biosciences | Cat#553294 | |
| IL-2 | Prometheus Laboratories | Cat#NDC65483-116-07 | |
| IL-12 | PeproTech | Cat#210-12 | |
| TGF-b1 | BioLegend | Cat#763104 | |
| IL-1b | R&D Systems | Cat#401-ML/CF | |
| IL-4 | PeproTech | Cat#214-14 | |
| Anti-IL-4 | BioXCell | Cat#BE0045 | |
| Anti-IFNg | BioXCell | Cat#BE0055 | |
| Mouse CD4+ T-cell Isolation Kit | STEMCELL Technologies | Cat#19852 | |
| Mouse IFN-gamma DuoSet ELISA | R&D Systems | Cat#DY485 | |
| Mouse IL-4 DuoSet ELISA | R&D Systems | Cat#DY404 | |
| Mouse IL-9 DuoSet ELISA | R&D Systems | Cat#DY409 | |
| Foxp3/transcription factor staining buffer set | Fisher Scientific | Cat#00-5523-00 | |
| Anti-CD4-eFluor 450 | eBioscience | Cat#48–0042–82 | |
| Anti-CD25-PerCP | TONBO Biosciences | Cat#65-0251 | |
| Anti-Foxp3-PE | Fisher Scientific | Cat#12-5773-82 | |
| PMA | Sigma-Aldrich | Cat#P1585 |
Troubleshooting
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.
Protocol references
1. Liu X, Chen X, Zhong B, Wang A, Wang X, Chu F, Nurieva RI, Yan X, Chen P, van der Flier LG, Nakatsukasa
H, Neelapu SS, Chen W, Clevers H, Tian Q, Qi H, Wei L, Dong C. Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development. Nature. 2014 Mar 27;507(7493):513-8. doi: 10.1038/nature12910. Epub 2014 Jan 19. PMID: 24463518; PMCID: PMC4012617.
2. Xu T, Stewart KM, Wang X, Liu K, Xie M, Ryu JK, Li K, Ma T, Wang H, Ni L, Zhu S, Cao N, Zhu D, Zhang Y, Akassoglou K, Dong C, Driggers EM, Ding S. Metabolic control of TH17 and induced Treg cell balance by an epigenetic mechanism. Nature. 2017 Aug 10;548(7666):228-233. doi: 10.1038/nature23475. Epub 2017 Aug 2. PMID: 28783731; PMCID: PMC6701955.
3. Lu Y, Wang Q, Xue G, Bi E, Ma X, Wang A, Qian J, Dong C, Yi Q. Th9 Cells Represent a Unique Subset of CD4+ T Cells Endowed with the Ability to Eradicate Advanced Tumors. Cancer Cell. 2018 Jun 11;33(6):1048-1060.e7. doi: 10.1016/j.ccell.2018.05.004. PMID: 29894691; PMCID: PMC6072282.
4. Xue G, Zheng N, Fang J, Jin G, Li X, Dotti G, Yi Q, Lu Y. Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to eliminate antigen-loss-variant tumor cells. Cancer Cell. 2021 Dec 13;39(12):1610-1622.e9. doi: 10.1016/j.ccell.2021.09.011. Epub 2021 Oct 21. PMID: 34678150; PMCID: PMC8678313.