Percp cy5.5 conjugated anti cd3

Cells from the BALF were stained with surface markers, including PE-Cy7 conjugated anti-CD45 (BD Biosciences, CA, USA), AF488 conjugated anti-CD11b (BD Biosciences, CA, USA), and PE conjugated anti-Ly6G (BD Biosciences, CA, USA). Cells from blood and spleen were stained with various surface markers, including PE-Cy7 conjugated anti-CD45 (BD Biosciences, CA, USA), Percp-cy5.5 conjugated anti-CD3 (BD Biosciences, CA, USA), FITC conjugated anti-CD4 (BD Biosciences, CA, USA), PE conjugated anti-CD25 (BD Biosciences, CA, USA), and APC conjugated anti-CD39 (BD Biosciences, CA, USA).
Peripheral blood mononuclear cells (PBMCs) from ARDS patients and healthy donors were stained with PerCP conjugated anti-CD3 (BD Biosciences, CA, USA), FITC conjugated anti-CD4 (BD Biosciences, CA, USA), APC conjugated anti-Foxp3 (BD Biosciences, CA, USA), and PE conjugated anti-CD39 (BD Biosciences, CA, USA). This study was approved by the Jinling Hospital Ethics Review Committee and written informed consent was provided by all subjects or their legal representatives.

Peripheral white blood cells were isolated through hemolysis by adding FACS lysing solution (BD Biosciences, San Jose, CA, USA). After the precipitates were washed twice by phosphate-buffered saline (PBS), cells were labelled according to our routine method [14 (link)]. Antibodies for PerCP-Cy5.5-conjugated anti-CD3; APC-conjugated anti-CD4; FITC-conjugated anti-CD45RA; PE-conjugated anti-CD25 and anti-Vδ2; PE-Cy7-conjugated anti-CD28 and anti-NKG2D; BV421-conjugated anti-56, anti-127, anti-CD194, and anti-TCRγδ; BV510-conjugated anti-CD8 and anti-NKP46; Alexa Fluor 647-conjugated anti-CCR7 and anti-CXCR5; Alexa Fluor 484-conjugated anti-CD183, and BB515-conjugated anti-PD-1 were purchased from BD Biosciences.
Samples were run on a BD LSR Fortessa™ cell analyzer (BD Biosciences) at Shuangzhi Purui Medical Laboratory Co., Ltd. (China), and the data were analyzed using FlowJo 10.1 software (Tree Star Inc., Ashland, USA).

All antibodies were pre-titrated to determine appropriate working concentrations. Immunostaining with periridin-chlorophyll protein (PerCp)-Cy5.5-conjugated anti-CD3 (BD Biosciences, clone UCHT1), allophycocynanin (APC)-H7-conjugated anti-CD8 (BD Biosciences, clone SK1) and APC-conjugated anti-CD161 (BD Biosciences, clone DX12) in the phenotypic characterization of MAIT cell was performed according to the protocol set by the commercial manufacturer (BD Biosciences). Surface staining for specific receptors was performed using monoclonal antibodies (mAbs) directed against: FITC-conjugated PD-1 (clone MIH4), phycoerythrin (PE)-conjugated CCR6 (clone 11A9), PerCp-Cy5.5-conjugated CCR5 (clone 3A9) and PE-conjugated TCR Vα7.2 (clone 3C10) (all mAbs procured from BD Biosciences). Immunostained samples were washed twice prior to acquisition on a FACS Canto II Immunocytometry system (BD Biosciences) and analyzed using FACS Diva software. Data were analyzed using FlowJo (version 9.3.1 and version 10). Doublets were excluded based on FSC-H and FSC-A, lymphocytes were identified based on FSC and SSC, and dead cells were excluded based on BD Horizon Fixable Viability Stain 510 (BD Biosciences). CD3⁺ and CD8⁺ T lymphocytes were identified. Fluorescence minus one (FMO) controls was used for optimal gating.