Anti cd3 percp

Briefly, for each sample, anticoagulant blood (heparin sodium salt) was treated with the lysing solution（Merck）to remove red blood cells. The solution was centrifuged. And the bottom cells were stained with 0.3 μl of anti-CD3-PerCP (Invitrogen, Austria) anti-CD4-FITC (Biolegend, USA), anti-CD8-APC (Biolegend, USA), and 30 μl of conjugated moAbs. After gently mixing and incubating for 30 min at 4 °C in the dark, the supernatant was removed. Then samples were washed with 800 μl of PBS and centrifuged at 700×g for 5 min. After adding 0.5 ml of PBS containing 1% paraformaldehyde, samples were analyzed by flow cytometer (Beckman Coulter, USA).

The reagents included bleomycin (Nippon Kayaku Ltd., Tokyo, Japan), saline (Kelun Pharmaceutical Co., Sicuan, China), Pirfenidone (Beijing Continent Pharmaceuticals Co., Ltd, Beijing, China), Hydroxyproline assay kit instructions (#A030-2-1, Nanjing Jian Chen Bioengineering Institute, China), protease inhibitor (Roche Life Science, USA), BCA protein assay kit (BIO-RAD, USA), polyvinylidene fluoride (PVDF) membranes (Millipore, Germany), skim milk (Beyotime, China), chemiluminescence detection kit (SuperSignal™ West Pico Chemiluminescent Substrate, Thermo Scientific, USA), enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen, Austria), lysing solution (Merck, Germany). Primary antibodies used in our project were as follows: α-Smooth Muscle Actin (Cell Signaling Technology, #19245, USA), anti-collagen I (Cell Signaling Technology, #81375, USA), β-Actin or glyceraldehyde-3-phosphate dehydrogenase (Cell Signaling Technology, #5174, USA), anti-CD3-PerCP (Invitrogen, Austria) anti-CD4-FITC (Biolegend, USA), anti-CD8-APC (Biolegend, USA). All horseradish peroxidase–conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

We used a FACS Calibur instrument (BD) and FlowJo software for all flow cytometric analyses (>10,000 events). In all cases, negative controls included isotype antibodies. Cells were washed once with PBS before the addition of antibodies. After 30 mins of incubation at 4°C in the dark, the cells were washed once and resuspended in FACS buffer (eBioscience, USA) before analysis.
T cells were analyzed with anti-CD8 PE, anti-CD4 APC, and anti-CD3 PerCP (eBioscience, USA), and tumor cell lines were analyzed with anti-LMP1 F(ab)₂ and anti-Fab FITC (Jackson ImmunoResearch, USA). CAR vectors also coded ZsGreen fluorescent protein, which can be detected by flow cytometry. The percentage of T cells positive for ZsGreen fluorescent protein indicated the lentivirus transfection efficiency. The surface expression of CAR was confirmed using APC anti-human IgG Fc antibody (BioLegend) and R-PE-protein L to detect scFv expression of CAR (Celltechgen technology) as previously described.15 (link)