Fitc conjugated anti cd45ra

Fluorescein (FITC)-conjugated anti-CD25 (catalog number 555431), phycoerythrin (PE)-conjugated anti-CD132 (catalog number 555900), allophycocyanin (APC), peridinin-chlorophyll proteins (PerCP)- and Pacific Blue (PB)-conjugated anti-CD4 (catalog numbers 555349, 347324 and 558116, respectively), PerCP- or FITC-conjugated anti-CD8 (catalog numbers 347314 and 555634, respectively), Alexa Fluor 647-conjugated anti-CD127 (catalog number 558598), FITC-conjugated anti-CD45RA (catalog number 555488), PE-conjugated anti-phosphorylated STAT5 (612567), PE Cy7-conjugated anti-PD-1 (catalog number 561272), Alexa Fluor 488-conjugated anti-IFN-γ (catalog number 557718) and Fixable Viability 510 (564406) were purchased from BD Biosciences. PE-conjugated anti-Bcl-2 (MHBCL04) was purchased from Thermo Fisher Scientific. Cell samples were acquired on a FACS Aria II flow cytometer (BD, USA) and analyzed with FlowJo software (Tree Star, San Carlos, CA, USA). Lymphocytes were gated based on their forward scattering and side scattering parameters, followed by the use of forward scatter area vs. forward scatter height dot-plot for doublet discrimination. The subsequent analyses were performed on viable cells (FV510^—) (S1A Fig)

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).

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll centrifugation. The following monoclonal antibodies (mAbs) and reagents were used in this study: PE-Cy7-conjugated anti-CD3, APC-conjugated anti-CD3, APC-Cy7-conjugated anti-CD4, PE-conjugated anti-CD38, APC-conjugated anti-HLA-DR, APC-Cy7-conjugated anti-HLA-DR, FITC-conjugated anti-CD45RA, PerCP-Cy5.5-conjugated anti-CCR7 (BD Biosciences, USA); Violet-conjugated anti-CD38, FITC-conjugated anti-CD38, PE-Cy7 conjugated anti-CD25, APC conjugated anti-CD69, APC conjugated anti-CD127, Amcyan-conjugated anti-CD45RA (BioLegend, San Diego, CA, USA). For the expression of all markers, flow cytometric gating was defined using fluorescence minus one (FMO) controls. CD4⁺ T cell subsets were identified in terms of CD45RA and CCR7 expression. CD38 and HLA-DR were measured on gated CD4⁺ T cell subsets: naive CD4⁺ T cells (Tn, CD3⁺CD4⁺CD45RA⁺CCR7⁺), central memory CD4⁺ T cells (Tcm, CD3⁺CD4⁺CD45RA⁻CCR7⁺), and effector memory CD4⁺ T cells (Tem, CD3⁺CD4⁺CD45RA⁻CCR7⁻). The expression of CD25, CD69, and CD127 were measured on gated CD38⁺ Tcm, HLA-DR⁻ Tcm, and CD4⁺HLA-DR⁺ cells. Data were collected using a BD LSRII flow cytometer (BD Biosciences) and analyzed using Flowjo software (TreeStar, USA).