Anti cd68 fitc

PHH were thawed, washed once with PBS and immediately fixed with 4% paraformaldehyde for 30 minutes on ice. After fixation, the cells were pelleted and blocked for 30 minutes on ice in PBS supplemented with 5% normal goat serum, 5% normal rabbit serum and 0.1% BSA for staining of cell-surface markers, or according to the manufacturer’s instructions for the eBioscience Intracellular Staining Kit (eBioscience, San Diego, CA) when detecting intracellular antigens. Next, cells were incubated with anti-CD45-FITC, anti-CD81-APC or anti-CD68-FITC (all BD Biosciences, San Jose, CA), anti-HLA-DR-FITC or anti-CD299-APC (both eBioscience), anti-human albumin-FITC (Rockland, Limerick, PA) or isotype-matched controls for 60 minutes on ice. All antibodies were used at manufacturer recommended concentrations except for anti-human albumin-FITC, which was used at 10 μg/mL. After staining, PHH were washed three times with PBS before being assessed by flow cytometry using a Fortessa LSR instrument (BD Biosciences). All flow cytometry data was analyzed using FlowJo V10.08r1 software (TreeStar Inc., Ashland, OR). A detailed description of all antibodies used is provided in S6 Table.

Collected 200 μl BM specimens from MM patients and HC, respectively. Subsequently, labeling the specimens with anti‐CD138‐Bv421, anti‐CD38‐APC, anti‐CSF1‐APC, anti‐CD14‐PerCP, anti‐CD68‐FITC, and anti‐CSF1R‐Bv421 antibodies was done (BD Biosciences). Among these, CD68 is an intracellular marker. The marker of intracellular staining was fixed and permeated with IntraSure Kit (BD Biosciences). Eventually, the data of CSF1 and CSF1R were acquired by flow cytometry (FCM) (Beckman CytoFLEX).