Cd8 percp clone sk1

Materials were from Sigma Aldrich (St. Louis, Mo, USA) except rK39 immunochromatographic test strips (InBios International, Seattle, WA, USA), anti-human CD4 (clone 4B12, Novocastra, Newcastle upon Tyne, UK,), Chemokine Receptor 4 (CCR4, clone 1G1), CD3 Peridinin chlorophyll (PerCP, clone SK7), CD8 Alexa Fluor 488 (clone RPA-T8), CD8 PerCP (clone SK1), CCR4 Phycoerythrin (PE, clone 1G1, BD Biosciences, San Jose, CA, USA), PD-1(clone EH12.2H7) and CD127 (clone A019D5) APC (Biolegend, San Diego, CA, USA), IL-5 (clone 9906, R&D Systems, Minneapolis, MN, USA), CCL17 (clone N-20), IL-10 (clone E-10), PD-1 (clone C-20), Perforin H (clone H-315), Granzyme B (clone 2C5, Santa Cruz Biotechnology, Dallas, TX, USA), antibody diluent, Target Retrieval Solution Citrate pH 6 (S2369), secondary detection system EnVision™ G|2 System/AP-Rabbit/Mouse (Permanent Red), EnVision™ FLEX Target Retrieval Solution, EnVision™ DuoFLEX Doublestain System (Dako, Glostrup, Denmark), RNALater and RNAqueous kit (Ambion, Austin, TX, USA), One-step reverse transcription kit from Qiagen (Hilden, Germany) and Bio-Plex Pro™ Human Chemokine Panel 40-Plex (BioRad, Hercules, CA, USA).

First, cells (around 10⁵ cells) were stained with the cell surface markers CD103 (αE)-FITC, Clone Ber-ACT8, BD Pharmingen) and CD8-PerCP (clone SK1, BD Biosciences). Then, cells were fixed in a fixation/permeabilization solution (eBioscience) for 10 minutes. After washing in a permeabilization solution (eBioscience), cells were incubated with mAb anti-granzyme B-PE (2 µg/ml, clone CLB-GB11, Sanquin, Amsterdam, The Netherlands) in the permeabilization solution (eBioscience) for 30 minutes. Cells were washed with PBS2+ and FACS analysis was performed.

pL-CRISPR.SFFV.GFP(Addgene plasmid # 57827) was a gift from Benjamin Ebert^{47 (link)}. pMD2.G (Addgene plasmid # 12259) and psPAX2(Addgene plasmid # 12260) were gifts from Didier Trono. EX-U0767-Lv105 for overexpressing PD-L1 was purchased from GeneCopoeia. PD-1 (APC, clone MIH4), PD-L1 (APC, clone MIH1), IFNγ (PE-Cy7, clone B27), CD107a (PE, clone H4A3), CD3 (APC-H7, clone SK7), CD4 (PerCP, clone SK3), CD8 (PerCP, clone SK1) and CD8 (BV650, clone SK1) antibodies were purchased from BD Biosciences; IL-2 (BV421, clone MQ1-17H12) was purchased from BioLegend. TNFα (APC, clone MAb11) was purchased from eBioscience.