Apc conjugated rat igg2b

Splenocytes were collected from glycolipid‐treated mice and diluted with staining solution (PBS (pH = 7.4) containing 0.1% bovine serum albumin). For further analysis of cell subpopulations, an array of antibodies was used for surface staining. All fluorescence‐conjugated antibodies were obtained from BioLegend as follows: FITC‐conjugated CD45 (30‐F11), PE‐conjugated antimouse CD54 (YN1/1.7.4), APC‐conjugated CD3 (145‐2C11), APC/Fire750‐conjugated CD8 (53–6.7), FITC‐conjugated CD4 (GK1.5), Brilliant Violet (BV) 421‐conjugated F4/80 (BM8123137), BV605‐conjugated CD11b (M1/70), APC/Fire750‐conjugated CD11c, PE‐conjugated NK1.1 (PK136), BV510‐conjugated Ly‐6C (HK1.4), and BV785‐conjugated Ly‐6G (1A8). After blocking with TruStain fcX (antimouse CD16/32) at 4 °C for 15 min, 2 × 10⁶ cells were stained with antibodies at 4 °C for 30 min, using 0.2 µg antibody per sample in 50 µL staining solution.
Analogously, isotypes were also applied in FACS analysis, including APC‐conjugated Armenian Hamster IgG, FITC‐conjugated rat IgG2b, APC/Fire750‐conjugated rat IgG2a, PE‐conjugated rat IgG1, PE‐conjugated rat Ig2b, PE‐conjugated mouse IgG2a, APC/Fire750‐conjugated Armenian Hamster IgG, FITC‐conjugated rat IgG2b, APC‐conjugated rat IgG2b, PE‐conjugated rat IgG2a, and PE/cy7‐conjugated rat IgG2b.k (Biolegend).

Blood samples were collected from anaesthetized mice 1 h after histone injection (45 μg/g). To prevent coagulation, blood was mixed with 3.13% (w/v) sodium citrate. Direct immunofluorescence staining was performed according to a previous report41 (link) with a slight modification. In brief, blood (25 μL) was added to the microcentrifuge tube. To identify PLAs, the blood was mixed with monoclonal antibodies (Abs) against CD45, CD11b, and CD41. The antibodies were as follows: APC-conjugated rat IgG_2b anti-mouse CD45 (clone 30-F11; Biolegend, San Diego, CA), PE-conjugated rat IgG_2b anti-mouse CD11b (clone M1/70; Biolegend), and BV421-conjugated rat IgG₁ anti-mouse CD41 (clone MWReg30; Biolegend) Abs. The isotype controls were APC-conjugated rat IgG_2b (Biolegend), PE-conjugated rat IgG_2b, and BV421-conjugated rat IgG_1. This mixture was gently stirred without vortexing and then incubated for 15 min at room temperature (18–25 °C) in the dark. Subsequently, the blood was fixed by adding lysing solution (Becton Dickinson, San Jose, CA) and incubation in the dark for 30 min. FACS analysis was performed using a BD LSR Fortessa X-20 system (Becton Dickinson) to detect expression changes on the cell surfaces.