Cd71 apc

To quantify CD4⁺ICOS⁺CD38⁺ and CD8⁺ICOS⁺CD38⁺ T cells, PBMCs were first resuspended with Human TruStain Fcx (Biolegend) for 10 min at room temperature and then stained with the following antibodies in FACS buffer (PBS + 2% fetal bovine serum): CD8a e450 (Invitrogen 48-0086-42, 1:200) ICOS BV605 (Biolegend 313538, 1:50), CCR7 PE (Biolegend 353204, 1:200), CD38 PerCP (Biolegend 303520, 1:100), CD4 PECy7 (Biolegend 357410, 1:100), and CD3 APCCy7 (Biolegend 300318, 1:200). Tfh markers consisted of CXCR5 APC (Biolegend 356907, 1:200) and PD-1 PE (Biolegend 135205, 1:100). CD71 APC (Biolegend 334108, 1:100) was also measured on sorted cells. Cells were analyzed on a Miltenyi MACSQuant16 Analyzer with single-stain control PBMC samples used for compensation conducted in FlowJo v10.6.2.

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).