Fusion flow cytometer

Nuclei suspensions were stained with 10 μg/mL 7-AAD (Invitrogen, Cat# A1310) and sorted on a BD Fusion Flow Cytometer (BD FACSMelody4-Way Cell Sorter) with a 100 µm nozzle. Nuclei were counted using a Countstar Automated Cell Counter (Countstar Rigel S5) and approximately 20,000 nuclei per sample were loaded into the 10x Chromium system using the Single-Cell 3′ Reagent Kit v3.1 according to the manufacturer’s instructions (10x Genomics). GEM-Reverse Transcription was performed with a thermal cycler using the following program: 53℃ for 45 min, 85℃ for 5 min, and held at 4℃. After reverse transcription and cell barcoding, emulsions were lysed and cDNA was isolated and purified with Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Scientific), followed by PCR amplification. The cDNA was then evaluated using an Agilent Bioanalyzer. Enzymatic fragmentation and size selection were applied to optimize the cDNA amplicon size. Then, the P5, P7, i7, and i5 sample indexes and TruSeq Read 2 (read 2 primer sequence) were added by end repair, A-tailing, adaptor ligation, and PCR. The final libraries containing the P5 and P7 primers were sequenced by an Illumina NovaSeq 6000 sequencer with 150 bp paired-end reads, aiming for a coverage of approximately 50,000 raw reads per nucleus.

The samples were incubated with red blood cell lysis buffer for 10 min at 4°C, then incubated with the appropriate antibodies (Table 1) at standard concentrations for 30 min. After being washed three times with phosphate‐buffered saline (PBS) and centrifuged, the cells were resuspended in 300 μL of PBS and subjected to fluorescence activated cell sorting (FACS). Data were acquired on a BD Fusion flow cytometer and analysed using FlowJo software.
Apoptosis was assessed by flow cytometry. The blood cells were harvested, washed twice with PBS, and resuspended in binding buffer. An Annexin V‐FITC/PI Apoptosis Detection Kit was used according to the manufacturer's instructions. Then, cells were harvested by centrifugation at 400 g for 5 min, resuspended in 400 μL of PBS, and analysed using a BD Fusion flow cytometer.
Quantification of absolute cell number was performed by flow cytometry. Peripheral whole blood from tail bleeds was lysed by red blood cell lysis buffer for 10 min at 4°C. Precision count beads (100 μL per sample) were used to calculate the cell population of each lineage. The beads were mixed with cells in PBS for 50 μL per sample before flow cytometry analysis. Absolute cell counts per microliter were calculated based on the precision count beads.