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3 protocols using pe cy 7 anti ccr7

1

Quantification of Circulating Memory Tfh Cells

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Human PBMCs at 106/tube were stained in duplicate with allophycocyanin (APC)-H7-anti-CD3, BV510-anti-CD4, fluorescein isothiocyanate (FITC)-anti-CD45RA, phycoerythrin (PE)-Cy7-anti-CCR7, peridinin-chlorophyll proteins (PerCP)-Cy5.5-anti-CXCR5, PE-anti-ICOS, BV421-anti-PD-1, PE-CF594-anti-CD154, or proper IgG isotype controls (Becton Dickinson, San Diego, CA, USA) at room temperature for 30 minutes. After being washed with phosphate-buffered saline (PBS), the cells were analyzed by flow cytometric analysis using a BD FACSAria II (BD Biosciences, San Jose, CA, USA). The data were analyzed with FlowJo software (version 7.6.2, by Flowjo LLC, OR, USA). We analyzed at least 50,000 events per sample and calculated the numbers of different subsets of circulating memory Tfh cells in individual samples according to the counts of lymphocytes per liter of blood multiplied by the percentage of different subsets of memory Tfh cells in lymphocytes.
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2

Characterization of Memory T Follicular Helper Cells

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Human PBMCs at 106/tube were stained in duplicate with APC-H7-anti-CD3, BV510-anti-CD4, FITC-anti-CD45RA, PE-Cy7-anti-CCR7, PerCP-Cy5.5-anti-CXCR5, PE-anti-ICOS, BV421-anti-PD-1, PE-CF594-anti-CD154, or isotype-matched control IgG (Becton Dickinson, San Diego, USA) at room temperature for 30 minutes, respectively. After being washed with PBS, the cells were subjected to flow cytometry analysis in a BD FACSAria Ⅱ (BD Biosciences, San Jose, CA USA) and data were analyzed with FlowJo software (v7.6.2). At least 50,000 events per sample were analyzed. The cells were gated initially on living lymphocytes, on CD3+ and CD4+, and then on CXCR5+ and CD45RA-. Subsequently, the frequency of ICOS+, PD-1+ or CD40L+ CD3+CD4+CXCR5+CD45RA- T (memory Tfh) cells were analyzed. In addition, the CD3+CD4+CXCR5+CD45RA- Tfh cells were gated on CCR7 and the frequency of ICOS+, PD-1+, CD40L+ CCR7+ or CCR7- Tfh cells was analyzed. The numbers of each type of memory Tfh cells in individual subjects were calculated by the counts of lymphocytes per liter of blood multiplied by the percentage of different subsets of memory Tfh cells in lymphocytes.
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3

Multicolor Flow Cytometry of T-cell Subsets

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Multicolor flow cytometry was performed on fresh PBMCs and frozen LNMCs. T-cell subpopulations were determined using the different combinations of the following fluorochrome-conjugated antibodies: Pacific Blue, PE-Cy7 or Alexa-Fluor 700anti-CD3, PerCP or Alexa-Fluor 700 anti-CD4, PE anti-CD27, PE-Cy7 anti-CCR7, PE-CF594 anti-CD45RO, Alexa-Fluor 488 anti-CXCR5, Alexa-Fluor 647 anti-CCR4, PE anti-CXCR3, PE-Cy7 anti-CCR6 (Becton Dickinson Biosciences, Pont de Claix (Le), France). Vioblue anti-CD3, APC-Vio770 anti-CD4, APC-Vio770 anti-CD45RO, APC anti-CD28 (Milteniy Biotech, Paris, France). Brillant Violet 421 anti-CD279 (Biolegend, London, UK).
Intracellular staining was carried out using the FoxP3 permeabilization solution kit according to the manufacturer's instructions (eBioscience, Paris, France) together with PE Ki67, PE anti-Bcl-6 (BD Pharmingen) or PE anti-FoxP3 (Biolegend), and Alexa-Fluor 488 anti-SAMHD1 (a gift from O. Schwartz) or rabbit anti-SAMHD1 (Euromedex, Souffelweyersheim, France) followed by Alexa-Fluor 647 antirabbit antibody (Invitrogen, Life Technologies, Saint Auben, France).
Dead cells were excluded using the Live/Death Vivid detection kit labeled with an aqua dye (Invitrogen). Fluorescence intensities were measured with an LSR II flow cytometer (Becton Dickinson) and analyzed using FlowJo version 7.6.5 (Tree Star Inc., Ashland, Oregon, USA).
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