Anti human cd235a fitc

BM, spleen (SPL), and peripheral blood (PB) were obtained from mice. Single-cell suspensions were prepared from each tissue following standard procedures. Erythrocytes were hemolyzed using BD Pharm Lyse buffer (BD Biosciences). Leukocytes were stained with fluorochrome-conjugated human antibody/mouse antibody for 20 min at room temperature in FACS buffer. After washing the labeled cells in 1x PBS, the cells were re-suspended in FACS buffer. Fluorescent signals were detected with a FACSVerse^TM flow cytometer, and data were analyzed using FlowJo software (BD Biosciences). The following human antibodies were used to measure human engrafted hematopoietic cells and mouse hematopoietic cells: anti-human CD45-APC and anti-human CD66b-FITC were purchased from Beckman Coulter. Anti-mouse CD45-FITC, anti-human CD33-FITC, anti-human CD14-PE, anti-human CD11b-PE, anti-human CD41-PE, anti-human CD235a-FITC, anti-human CD3-FITC, anti-human CD19-APC were purchased from BioLegend.

For measurement of mCherry fluorescence intensities, 3.0 × 10⁵ cells were resuspended in FACS buffer (PBS containing 2% FBS), filtered through a cell strainer and analyzed using a BD LSRFortessa Cell Analyzer (BD Biosciences) with BD FACS Diva software (BD Biosciences).
For the confirmation of erythroid differentiation, 1 × 10⁵ cells were stained with anti-human CD235a-FITC (#349103, Biolegend, 1:20) and CD71-APC (#334107, Biolegend, 1:20) in a total volume of 10 µL FACS buffer containing 0.5 µL Human TruStain FcX^TM (biolegend). Isotype controls were conducted using FITC and APC Mouse IgG2a antibodies (#400209 and #400221, Biolegend). After a 20 min incubation on ice, the samples were washed with 100 µL PBS containing 0.5 µg/mL DAPI. Finally, the cells were resuspended in 100 µL FACS buffer and passed through a cell strainer for analysis on a BD FACS ARIA II Cell Sorter (BD Biosciences) with the BD FACS Diva software version 8.0.1 (BD Biosciences).
For measurement of PPIX fluorescence intensities, which displays an autofluorescence with a peak at 632 nm when excited at 409 nm^{57 (link)}, the Qdot^® 605 filter setting (excitation 405 nm, emission 610 nm + /− 20 nm) of the BD FACS ARIA II Cell Sorter was used.
Flow cytometry data were analyzed by FlowJo software v9.7.6 or higher (Tree Star).