Pe conjugated rat igg2a

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).

The surface markers CCR7 (M1) and CD206 (M2) were examined via flow cytometry for evaluating different macrophage phenotypes. The specific procedure is the same as described in previous studies [5 (link)]. Anti-mouse CD16/32 (Biolegend, USA) was used to block the non-specific antigens. The antibodies for flow cytometry in this study included PE-conjugated CCR7 and PerCP-conjugated CD206 (Biolegend, USA), and the isotype controls were PE-conjugated Rat IgG2a, ĸ and PerCP-conjugated Rat IgG2a, ĸ (Biolegend, USA). The expression level of M1/M2 macrophage markers of all samples was analyzed using a flow cytometer (NovoCyte, ACEA Biosciences, USA) in triplicate. In addition, the surface markers IL-1β (M1) and CD163 (M2) were detected via western blot for further evaluating different macrophage phenotypes. The first antibodies for western blot were anti-IL-1β (ABclonal, Wuhan, China, the dilution was 1:1000), anti-CD163 (ABclonal, the dilution was 1:1000) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ABclonal, the dilution was 1:4000).