TH17 polarization protocol Th17细胞体外分化

Materials Buffers/Media
Balb/c mouse splenocytes, 3x10 per 6 well culture plate Anti-mouse CD3, 1 μg/ml (clone: 145-2C11, cat# 16-0031) Anti-mouse CD28, 10 μg/ml (clone:37.51, cat# 16-0281) Anti-mouse IL-2, 1 μg/ml (clone: JES6-1A12, cat# 16-7022) Anti-mouse IL-4, 5 μg/ml (clone: 11B11, cat# 16-7041) Anti-mouse IFN, 5 μg/ml (clone: XMG1.2, cat# 16-7311) Recombinant human TGF-β, 1ng/ml (cat# 14-8348) Recombinant mouse IL-6, 40ng/ml (cat# 14-8061) Recombinant mouse IL-23, 40 ng/ml (cat# 14-8231) PMA , 50 ng/ml (Phorbol 12-myristate 13-acetate, Sigma cat# P7585) Ionomycin, 1 μg/ml (Sigma cat# I0634) Brefeldin A solution, 1:1000 (cat# 00-4506) 6 well culture plates T-25 culture flasks
Mouse Th17 Polarization Page 3 of 3
Mouse Th17 Polarization Protocol
Research Use Only
6.
On Day 9-10, treat for 5 hours with: PMA (50ng/ml) (1:2000) and Ionomycin (1ug/ml) (1:1000) in the presence of Brefeldin A (1:1000). Also treat some cells with Brefeldin A (1:1000) alone as a control
7.
Cells are ready for the relevant staining protocol.
Revised 8-31-2010 Provided as a courtesy by eBioscience, Inc. Copyright © 2000-2010 eBioscience, Inc. Tel: 888.999.1371 or 858.642.2058 • Fax: 858.642.2046 • www.ebioscience.com • info@ebioscience.com
Mouse Th17 Polarization Page 2 of 3
Mouse Th17 Polarization Protocol
Research Use Only
Experimental Procedure
1. Pre-coat 6 well TC plates with 2 ml of anti-mouse CD3 (1 μg/ml in PBS) (clone: 145-2C11). Incubate 1-2 hours at 37°C or overnight at 4°C for sufficient binding. Remove the PBS/anti-CD3 by pipetting just prior to adding the cells (no need to wash, though). Coating plates can be sealed with parafilm and stored at 4°C for <2 weeks with PBS/anti-CD3 the solution. Plate 5x10 splenocytes per well (0.5 ml of spleen) on the coated 6-well plate in 3 ml cIMDM or cRPMI with: anti-mouse CD28 (10 μg/ml) anti-mouse IL-2 (1 μg/ml) anti-mouse IL-4 (5 μg/ml) anti-mouse IFNg (5 μg/ml) recombinant hTGF-b (1 ng/ml) recombinant mIL-6 (40 ng/ml) It is easiest to mix the antibodies and cytokines into the medium in bulk, then add the cells and divide over the number of wells. (example: 3 ml x 6 wells = 18 ml total 7 including 3x10 cells) Incubate 3 days at 37°C. On day 3, culture the cells for 3 more days in the presence of recombinant mouse IL-23 (cat. 14-8231) Harvest cells by pipetting up and down Centrifuge and remove supernatant Resuspend the pellet in cIMDM or cRPMI with 40 ng/ml recombinant mouse IL-23 (32 ml per harvested plate) Plate 8 ml per T-25 flask (one 6 well plate 4 T-25 flasks) Culture 3-4 more days On day 6-7, EITHER: a. treat for 5 hours with: PMA (50ng/ml) and Ionomycin (1ug/ml) in the presence of Brefeldin A (1:1000). Also treat some cells with Brefeldin A (1:1000) alone as a control OR b. Culture the cells for 3 more days in the presence of recombinant mouse IL-23 (cat. 14-8231) Harvest cells by pipeting up and down Centrifuge and remove supernatant Resuspend in cIMDM or cRPMI with 40 ng/ml recombinant mouse IL-23 Plate 8 ml per T-25 flask Culture 3-4 more days
Experiment Duration 10 day culture is recommended IL-17F, IL-21, IL-22, IL-17A 6 day culture is adequate for IL-17A only
Revised 8-31-2010 Provided as a courtesy by eBioscience, Inc. Copyright © 2000-2010 eBioscience, Inc. Tel: 888.999.1371 or 858.642.2058 • Fax: 858.642.2046 • www.ebioscience.com • info@ebioscience.com
7
Complete RPMI (10% FBS, penicillin-streptomycin, 2-ME) Complete IMDM (10% FBS, penicillin-streptomycin) eBioscience IC Fixation Buffer (cat# 00-8222) eBioscience 10X Permeabilization Buffer (cat# 00-8333)
6
2.
3. 4.
5.ຫໍສະໝຸດ Baidu
Revised 8-31-2010 Provided as a courtesy by eBioscience, Inc. Copyright © 2000-2010 eBioscience, Inc. Tel: 888.999.1371 or 858.642.2058 • Fax: 858.642.2046 • www.ebioscience.com • info@ebioscience.com
Mouse Th17 Polarization Page 1 of 3
Mouse Th17 Polarization Protocol
Research Use Only
General Notes
Note I: Cultures can be treated with PMA, Ionomycin and Brefeldin A for 5 hours and then fixed with IC fixation buffer. After fixation, wash with Flow Staining Buffer and store at 4C for up to 2 weeks. At time of staining, cells can be permeabilized by washing twice with 1X Permeabilization buffer and then stained per the relevant protocol. Staining of fixed and stored cells will likely be dimmer than freshly cultured cells.. Note II: Some publications suggest that IMDM may promote IL-22 expression better than RPMI. We have not seen much of a difference.