Purified rat anti mouse cd16 cd32 mouse fc block

BALB/c mice were first shaved to remove the hair on their back. The materials used included nanoreactors (100 μg), toxin (4 μg), heated toxin (4 μg, 70 °C inactivated for 1h), and nano-toxin (4 μg toxin absorbed by 100 μg nanoreactors), and PBS as control. These materials were injected subcutaneously on day 0, followed by a boost on day 7 and day 14. On day 21 after immunization, the lymph nodes were collected and dissociated into single cell suspensions for flow cytometric analysis. After staining the lymph nodes with APC Rat Anti-Mouse IgD (BD Pharmingen, 560868, 1:100), Alexa Fluor 488 Rat Anti-Mouse CD45R (BD Pharmingen, 557669, 1:100), PE Anti-mouse/human GL-7 Antigen (T- and B-cell Activation Marker) (BioLegend, 144607, 1:100), Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) (BD Pharmingen, 553142, 1:100) data were collected on a flow cytometer and analyzed using Flowjo software.

Nickel(II) Chloride was obtained from Wako Pure Chemical Corporation. Freund’s incomplete adjuvant (IFA) and complete adjuvant (CFA) were from MP Biomedicals. The primary antibody specific for Sema3A was from Abcam. Antibodies specific for phospho-p38 (Thr180/Tyr182) and p38 were obtained from Cell Signaling Technology. Rabbit antibody to GAPDH was obtained from Osenses. Rabbit antibody to β-actin was obtained from Bioss. Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody were obtained from Cell Signaling. FITC anti-mouse CD11c, Alexa Fluor 488 anti-mouse CD11b, PE anti-mouse F4/80, PE anti-mouse I-A/I-E, FITC anti-mouse CD3, PE/Cyanine7 anti-mouse CD4 and PE anti-mouse CD8a were purchased from BioLegend. APC-Cy™7 Rat Anti-Mouse CD45 and Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) were from BD Biosciences. 7-AAD Viability Staining Solution was from Invitrogen. Anti-rabbit Alexa Fluor 488 secondary antibody was from Abcam. Readidrop^TM Propidium Iodide was from Bio-Rad Laboratories. The recombinant mouse Sema3A were purchased from R&D Systems.

Mature adipocytes and stromal vascular fractions (SVF) were isolated as described below. Epididymal fat tissues were minced and incubated in a fresh digesting media of Krebs Ringer HEPES (KRH) buffer for 60 min at 37 °C and were separated into adipocytes and SVF by using mesh with a grid diameter 300 µm. The SVF cells were subjected flow cytometry analysis as previously described^{30 (link)}. The cells were first incubated at 4 °C for 10 min with Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block) (BD Pharmingen) to reduce nonspecific binding of antibodies to FcR receptors. The cells (1 × 10⁶) derived from epididymal fat tissues were incubated at 4 °C for 30 min in Stain Buffer (BD Pharmingen) with the relevant optimized amount of fluorochrome conjugated antibodies or the appropriate isotype controls: PerCP-Cy 5.5 Rat Anti-Mouse CD11b, BV421 Hamster Anti-Mouse CD11c, PE-Cy 7 Mouse Anti-Mouse CD45.2 (BD Pharmingen), Rat Anti-Mouse CD206 Alexa Fluor 647 (AbD Serotec), and Anti-Mouse F4/80 Antigen FITC (eBioscience). Dead cells were excluded from analysis using 7-aminoactinomycin D staining (BD pharmigen). All data were acquired with FACSAria I flow cytometer (BD Biosciences) and analysed using FlowJo software (TreeStar, Ashland, OR).