Anti fasl af647 antibody mlf4 clone

All the reagents used in this study for flow cytometry were purchased from Biolegend (San Diego, CA, USA) unless otherwise specified. MSC were labeled with the following antibodies: anti-mouse Sca1-PE, anti-mouse CD73-PE/Cy7, and anti-mouse CD45-PE. Splenocytes were stained with anti-mouse CD45-PE, anti-mouse CD4-BV785, and anti-mouse CD8a-APC/Fire750 antibodies. Apoptosis of splenocytes was evaluated by flow cytometry after labeling the cells with PI and Annexin V-APC according to manufacturer’s protocol. Engraftment efficiency was tested with anti-mouse CD45.1-PE and anti-mouse CD45.2-APC. The antibodies used for peripheral blood immunophenotyping were: anti-mouse CD45.1-PE, anti-mouse CD3-PacificBlue, anti-mouse CD4-BV785, anti-mouse CD8a-APC/Fire750, and anti-mouse CD25-APC. Treg were identified following labeling of splenocytes with the same antibodies as for immunophenotyping and anti-FoxP3-AlexaFluor488. Prior to FoxP3 staining, the cells were fixed and permeabilized using True-Nuclear Transcription Factor Buffer Set according to manufacturer’s protocol. FasL was detected using anti-FasL-AF647 antibody (MLF4 clone, Bio-Rad, CA, USA). For all the antibodies, we used the corresponding isotypes from the same sources. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, IN, USA) and CytExpert software, version 2.1.

For flow cytometric analysis, MSC were stained with the following antibodies: anti-mouse Sca1_PE, anti-mouse CD73_PE/Cy7, anti-mouse CD117_APC (c-kit), anti-mouse CD45_PE, anti-mouse CD44_PE, anti-mouse CD95_PE (Biolegend, San Diego, CA, USA), goat anti-mouse CD105 and the secondary rabbit anti-goat_FITC (Invitrogen, Carlsbad, CA, USA). The antibody mix that was used to label the splenocytes contained anti-mouse CD45_PE, anti-mouse CD4_BV784 and anti-mouse CD8a_APCfire (Biolegend, San Diego, CA, USA). FasL was detected using anti-FasL_AF647 antibody (MLF4 clone, Bio-Rad, CA, USA). For all the antibodies, we used the corresponding isotypes from the same sources. Samples were analyzed using a Cytoflex flow cytometer (Beckman Coulter, Indianapolis, IN, USA) and CytExpert software. Determination of the proliferation index of the splenocytes was done based on the carboxyfluorescein succinimidyl ester (CFSE) readings that were processed using ModFit LT^TM software (Verity Software House, Topsham, ME, USA). For the CFSE analysis, the unstimulated splenocytes were used as the parent (basal fluorescence).