Fitc labeled anti cd11b

Freshly isolated PBMCs and those treated with IronQ were collected in PBS and subsequently blocked for 30 min at 4 °C by using a blocking reagent (PBS containing 2.5% BSA). Cell suspensions were subjected to staining in PBS containing 0.1% BSA at a concentration of 5 × 10⁵ cells. Each 100 µL sample of the cell suspension was incubated with the appropriate IgG isotype controls for each fluorescence channel or with specific antibodies, including FITC-labeled anti-CD34 (Elabscience), FITC-labeled anti-CD14 (Miltenyi), FITC-labeled anti-CD11b (eBioscience), PE/Cy5.5‐labeled anti-CD45 (eBioscience), FITC-labeled anti-CD31 (Life Technologies), PE-labeled anti-CD105 (eBioscience), PE/Cy5.5‐labeled anti‐CD3(clone: UCHT1, Merck Millipore), APC‐labeled anti‐CD206 (clone 15–2, Sigma-Aldrich), APC‐labeled anti‐CCR2 (CD192, Sino Biological), PE‐labeled anti‐CD8a (clone: RPA‐T8, Merck Millipore), PE‐labeled anti‐CD4 (clone: RPA‐T4, Merck Millipore), and Alexa Fluor‐488‐labeled anti‐CD25 (Clone: BC96, eBioscience). This incubation was performed at 4 °C in the dark for 30 min. After two washes containing 0.1% BSA with PBS, the cell suspension was subjected to analysis by using flow cytometry (Beckman Coulter, Epics XL-MCL, CA, USA). Data analysis was carried out by using the FlowJo10 software (Tree Star, Ashland, Oregon), and scattergrams were gated based on three different cell sizes.

BMDMs or BMDMs with GFP-transfected were stained with PE-labeled anti-F4/80 (eBioscience, San Diego, CA, USA) or FITC-labeled anti-CD11b (eBioscience, San Diego, CA, USA). Prepared HNPCs were resuspended in PBS at 4 °C and pre-incubated with TruStain FcX™ (anti-mouse CD16/32) antibody (BioLegend Way, San Diego, CA, USA) to minimize non-specific antibody binding, then HNPCs were stained with PE-labeled anti-F4/80 antibody (eBioscience, San Diego, CA, US), FITC-labeled anti-CD11b antibody (eBioscience, San Diego, CA, US), and anti-GFP antibody (Abcam, Cambridge, MA, USA) with secondary antibody Alexa Fluor^® 647-conjugated antibody (Abcam, Cambridge, MA, USA). FACS analysis was performed using a FACS Calibur^TM flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA). Data were analyzed with the FlowJo software version 10.0 (FlowJo, Ashland, OR, USA).