Anti human cd45 apc fire 750

The biological analysis was conducted on fresh peripheral blood mononuclear cells (PBMCs) derived from blood samples. To assess the subsets of ILC1, ILC2, and ILC3 in peripheral blood among patients with elevated uric acid (UA), a flow cytometry assay was carried out. Briefly, 1 × 10⁶ cells were suspended in 100 µL phosphate-buffered saline (PBS) prior to antibody incubation. The cells were then incubated with Zombie Aqua fixable viability dye (BioLegend, San Diego, CA, USA), anti-human-CD45-APC/Fire 750 (BioLegend), anti-human-c-Kit-PE/Cy7, anti-human-CRTH2-PerCP/Cy5.5, anti-human-Lineage cocktail (CD3/14/16/19/20/56)-FITC, and anti-human-CD127-BV421 (all from BioLegend) at room temperature for 30 min in the absence of light. Following a wash with 2 mL PBS, the cells were resuspended in 500 µL PBS and examined using flow cytometry (FACS Aria™ II, BD Bioscience, NJ, China). List mode data files were analyzed using FlowJo™ 10 software. Total lymphocytes were identified based on forward and side scatter properties. The innate lymphoid cell (ILC) gate was defined by CD45 + Lineage-CD127 + expression. ILC subtypes were further distinguished as ILC1s (c-Kit-CRTH2-), ILC3s (c-Kit-CRTH2 +), and ILC2s (c-Kit +).

Isolation of immune cells from fresh blood was conducted by adding 1 mL ACK lysis buffer per 100 μL of blood, and lysis was performed on ice for 5 min. The lymphocyte fraction was depleted of red blood cells by the lysis. The cells were resuspended in the staining buffer (Thermo Fisher Scientific) and counted, then labeled with the viability stain (Zombie Aqua, BioLegend) in PBS for 15 min at room temperature, washed, and incubated with anti-human CD45-APC-Fire750 (Clone HI30, Biolegend) and anti-human CD3-BV785 (Clone UCHT-1, Biolegend) for 1 h at 4 °C in the staining buffer (Thermo Fisher Scientific). The cells were further washed and fixed in the intracellular (IC) fixation buffer (Thermo Fisher Scientific) overnight at 4 °C. Human CD45⁺ CD3⁺ CAR-T cells were analyzed using the Attune NxT flow cytometer.