Evaluation of nitrotyrosine expression on the surface of CD8+ T cells

doi:10.1038/nprot.2007.239

Method Article

Evaluation of nitrotyrosine expression on the surface of CD8+ T cells

https://doi.org/10.1038/nprot.2007.239

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Biochemistry

Computational biology and bioinformatics

Biological techniques

CD8 T cells

nitrotyrosine

This protocol is used to evaluate the level of nitrotyrosine on the surface of CD8+ T cells after incubation with myeloid derived suppressor cells

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.)

Centrifuge with temperature control

DMI6000 inverted Leica TCS AOBS SP5 tandem scanning confocal microscope

LAS AF version 1.5.1.889 software suite

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.
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.
Wash the cells with PBS + 2% FBS twice, complete media is added and the dishes are kept on ice.
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.

48 hours

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

See accompanied manuscript

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Evaluation of nitrotyrosine expression on the surface of CD8+ T cells

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Evaluation of nitrotyrosine expression on the surface of CD8+ T cells

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Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

Associated Publications

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Terms of Service

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