Pe conjugated cd34

Mouse monoclonal antibodies against human band 3 and Kell were generated in our laboratory [3] (link), [20] (link), [21] (link). Commercial antibodies included BV421-conjugated CD45 (Catalog No. 563879, BD Biosciences, Franklin Lakes, NJ), PE-conjugated CD235a/GPA (Catalog No. 555570, BD Biosciences), APC-conjugated CD235a/GPA (Catalog No. 551336, BD Biosciences), PE-Cy7-conjugated CD123 (Catalog No. 25-1239-42, Invitrogen, Carlsbad, CA), APC-conjugated CD49d (Catalog No. 304307, BioLegend, San Diego, CA), PE-conjugated CD34 (Catalog No. 555822, BD Biosciences), APC-conjugated CD36 (Catalog No. 550956, BD Biosciences), FITC-conjugated CD36 (Catalog No. 555454, BD Biosciences), PE-conjugated CD49e (Catalog No. 103805, BioLegend), FITC-conjugated CD47 (Catalog No. 556045, BD Biosciences), PE-conjugated CD71 (Catalog No. 555537, BD Biosciences), AF647-conjugated CD147 (Catalog No. 562551, BD Biosciences), and Hoechst 33342 (Catalog No. 561908, BD Biosciences).

The surface markers of isolated cells were analyzed by flow cytometry analysis. Briefly, the isolated cells (1 × 10⁶ cells, passage 3) from the three dogs were, respectively, suspended in 100 μL phosphate-buffered saline (PBS) containing 10 μg/mL antibodies for PE-conjugated CD29 (303003, BioLegend, USA), PE-conjugated CD44 (103024, BioLegend, USA), PE-conjugated CD90 (561970, BD Biosciences, USA), PE-conjugated CD105 (bs-0579R-PE, Bioss, CHN), FITC-conjugated CD73 (bs-23233R-FITC, Bioss, CHN), PE-conjugated CD34 (559369, BD Biosciences, USA), and FITC-conjugated CD45 (11-5450-42, eBioscience, USA). After incubation for 30 min at 4°C, the cells were washed with PBS and then resuspended in 500 μL of PBS for analysis. As for CD11b, the isolated cells (1 × 10⁶ cells, passage 3) were suspended in 100 μL phosphate-buffered saline (PBS) containing 10 μg/mL CD11b (MA5-16604, eBioscience, USA). After incubation for 30 min at 4°C, the cells were washed with PBS and then labeled with goat antirabbit IgG (H + L) cross-adsorbed secondary antibody, FITC (F-2765, Invitrogen), at a dilution of 1 : 500 for 1 h at room temperature. Cell fluorescence was evaluated by flow cytometry using a DxP Athena™ flow cytometry system (Cytek) and analyzed with FlowJo 10 software (Tree Star, USA).

The immunophenotype was analyzed within the 4th and 5th passages by fluorescence-activated cell sorting (FACS). In brief, 5 × 10⁵ cells (in 50 μl of staining buffer (SB, Pharmingen Becton Dickinson)) were mixed with 10 μl of the following antibodies: PE-conjugated CD73 (BioLegend, San Diego, CA), FITC-conjugated CD90 (Santa Cruz Biotechnology, California), PE-conjugated CD34 (BD Biosciences, NJ), RPE-conjugated CD45 (BD Biosciences, NJ), RPE-conjugated CD14 (Serotec, Kidlington, UK), and APC-conjugated CD105 (BioLegend). Proper isotype controls for each antibody were used to discard unspecific binding. Cells were incubated for 20 min at room temperature in the dark and then fixed and analyzed using an Attune flow cytometer (Life Technologies, Carlsbad, CA). Data analysis was performed using FlowJo version 7.2.2 software (Tree Star Inc., Ashland, OR). Nonviable cells were excluded according to the side scatter vs. forward scatter parameters, and 5,000 events were acquired for each sample.

Cultured cells (passage 1) were trypsinized and stained with a panel of antibodies for fluorescence-activated cell sorting (FACS) analysis. Approximately, 1 × 10⁵ cells were re-suspended in phosphate buffered saline (PBS) and incubated with IgG block for 5 minutes to block non-specific binding. The following antibodies were used: AF-700 conjugated CD3 (BD BioSciences, USA), PE conjugated CD14 (BD, Immunocytometry, USA), APC conjugated CD19 (BD BioSciences, USA), PE conjugated CD34 (BD, BioSciences, USA), APC conjugated CD44 (BD, Pharmingen, USA), FITC conjugated CD45 (BD Pharmingen, USA), PE conjugated CD73 (BD Pharmingen, USA), AF-700 conjugated CD90 (Biolegend, USA) and APC conjugated CD105 (Biolegend, USA). Cells were stained for 30 minutes at 4°C with the antibodies. After washing, samples were analyzed on a LSR II flow cytometer (BD, USA) and at least 10,000 events were acquired for each population. Data acquisition and analysis were performed using FACS DIVA software (BD Biosciences, USA). Unstained cells were used to establish flow cytometer settings. Debris and cells/particles with auto-fluorescence were removed by using a threshold on the forward scatter.