Hladr pacific blue

PFMC or PBMC were stained with LIVE/DEAD yellow fixable dead cell stain kit (Life Technology, Eugene, OR) and with the following two monoclonal antibody staining panels: In Panel 1, cells were surface-stained by anti-CD3-PerCP, CD4-PE-Cy7, CD45RO-FITC, CD69-APC, CD38-AF700 (BD Bioscience, San Jose, CA), CD8-PE-TR (Invitrogen, Frederick, MD), CCR7-APC-Cy7 and HLADR-Pacific Blue (BioLegend, San Diego, CA). Cells were then fixed, permeabilized, and washed with Transcription Factor Buffer Set (BD Pharmingen) according to the manufacturer and stained with anti-HIV-1 p24-PE (KC57) (Beckman Coulter, Indianapolis IN). In Panel 2, monoclonal antibodies included: anti CD25-APC (BioLegend), CCR5-V450 and intracellular staining for Ki67-BV711 (both from BD Bioscience) in addition to anti-CD3, CD4, CD8, CCR7, CD45RO, and HIV-1 p24 as in panel 1. Gates were set using Fluorescent-Minus-One controls for each sample. T cells were identified as naïve (CD45RO-CCR7+), central memory (Tcm) (CD45RO+CCR7+), effector memory (Tem) (CD45RO+CCR7-), and terminally differentiated effector memory (TemRA) (CD45RO-CCR7-). Stained samples were analyzed by a LSRII cytometer (BD). Data were analyzed using FlowJo (Tree Star, Ashland, OR).

Samples were resuspended in FACS buffer (2% FBS, 0.1% sodium azide) and stained with the following mixtures of Biolegend antibodies: BDCA1-Percp/Cy5.5, CD14-APC, HLADR-Pacific Blue, CD3/19/56-FITC, CD123-PE/Cy7, and either FcεRIα-PE or mouse IgG_2b-PE, all at manufacturer's recommended concentrations. Some samples were stained with CD45-A700 to identify hematopoietic cells and CD203c-biotin (Biolegend, at manufacturer's recommended concentrations) followed by streptavidin-A647 (Invitrogen, at manufacturer's recommended concentration) to identify mast cells. Cells were stained with antibodies and propidium iodide (PI) (Biolegend) at 1∶400 for 15 minutes at 4 degrees, washed and spun at 1300 rpm, and resuspended in FACS buffer. At least 1×10⁶ cells were run on slow or medium speed on a BD LSRII machine and analyzed with Flowjo software.

To analyze cell-surface expression of typical MSC surface marker proteins, cells were detached, counted and labeled with the following anti-human antibody conjugates: CD44 - APC; CD73 - APC; CD90 - PE; CD14 - PerCp/Cy5.5; CD45 - PerCp/Cy5.5; CD31 - FITC; CD34 - FITC; CD19 - Pacific Blue; HLA-DR - Pacific Blue (all from Biolegend) and also CD105 PE (eBioscience, Inc., San Diego, CA, USA). The mouse isotype antibodies used as the respective controls were: Pacific Blue IgG1; Pacific Blue IgG2a; IgG1k PErCp/Cy5.5; IgG2a PerCp/Cy5.5; IgG1k PE; IgG1k APC and IgG1k FITC (all from Biolegend®, San Diego, CA, USA). A total of 10,000 labeled cells were acquired using a Gallios Flow cytometer (Beckman Coulter) and results analyzed with Kaluza software (Beckman Coulter, Inc., Carlsbad, CA, USA).