Evaluation of nitrotyrosine expression on the surface of CD8+ T cells
Lab/Group: Gabrilovich lab (H. Lee Moffitt Cancer Center)
Related Journal & Article Information
Journal: Nature Medicine
Introduction
This protocol is used to evaluate the level of nitrotyrosine on the surface of CD8+ T cells after incubation with myeloid derived suppressor cells
Materials
Reagents
Phosphate buffered saline (PBS)
ACK lysing buffer 8.29g NH4Cl (0.15M), 1g KHCO3 (10.0mM), 37.2mg Na2EDTA(0.1mM) Add 800ml H20 and adjust pH to 7.2-7.4, Add H20 to 1l. Filter sterilize and store at RT.
Rabbit IgG, NT, CD8, or and Gr-1 antibodies
Specific peptide SIINFEKL
Lysis Buffer
10 mM Tris-Hcl pH 7.4, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM PMSF, and 1:100 protease inhibitor cocktail (Sigma, MO).
Complete media: complete Media RPMI 1640 with Hepes, 10% FBS, 1% antibiotic
Poly-d-lysine coated glass bottom culture dish (MatTek Corp.)
Equipment
Centrifuge with temperature control
DMI6000 inverted Leica TCS AOBS SP5 tandem scanning confocal microscope
LAS AF version 1.5.1.889 software suite
Procedure
1. Culture splenocytes from OT-1 mice at 37 °C in a 95% air/5%CO2 saturated with H20 with SIINFEKL peptide (10 μg/ml) in the presence of MDSC (at 3:1 ratio) on a poly-d-lysine coated glass bottom culture dish for 5h, 24h or 48h in complete media.
2. Label the cells with 6 μl of antibodies against Gr-1 (conjugated with PE), CD8 (conjugated with Alexa 647), Nitrotyrosine (conjugated with Alexa 488), or isotype IgG conjugated with Alexa 488 and incubated on ice for 40-60 min.
3. Wash the cells with PBS + 2% FBS twice, complete media is added and the dishes are kept on ice.
4. View the cells with a DMI6000 inverted Leica TCS AOBS SP5 tandem scanning confocal microscope with a 40x /1.30NA oil immersion objective. Tunable 488 Argon and 546 and 633 laser lines were applied to excite the samples using AOBS line switching to minimize crosstalk between fluorochromes. Images and Z-stacks are produced with three cooled photomultiplier detectors and the LAS AF version 1.5.1.889 software suite.
Troubleshooting
Lyse all the red blood cells.
Make sure the purity of MDSC is 95% and above.
Increase the time of incubation or amount of antibodies if you see a weak signal.
PE fades faster than Alex-fluor dyes
Critical Steps
Anticipated Results
See accompanied manuscript
References
Acknowledgements
Keywords
CD8 T cells
nitrotyrosine