Anti cd16 32antibody

Spleens were prepared into single cell suspension and red blood cells were lysed
in cold distilled water. After filtered through 70-µm nylon mesh and counting
using a MACSQuant analyzer (Miltenyi Biotec), cells were incubated with anti-CD16/32
antibody (Bio X Cell) to block Fc receptors and stained with fixable viability dye eFluor
780 (eBioscience). Cells were then stained for 20 min in staining media (Hank’s
balanced salt solution, 3% FBS, 0.02% sodium azide, 1 mM EDTA) with
primary antibodies including B220-FITC, B220-eVolve 655 (clone RA3–6B2),
GL7-eFluor 660, GL7-eFluor 450 (clone GL7), CD95-PE, CD95-APC (clone 15A7) (eBioscience),
peanut agglutinin (PNA)-biotin (Vector Laboratories), CD86-Pe-Cy7 (clone GL-1; BioLegend)
or CD184-biotin (2b11/CXCR4; BD Biosciences). Cells stained with biotin-labeled antibodies
were incubated with streptavidin-eFluor 450 (eBioscience). For intracellular staining,
cells were stained with fixable viability dye eFluor 780, permeabilized and fixed using a
cytofix/cytoperm plus kit (BD Biosciences) according to manufacturer suggested protocol.
Cells were then stained with YY1 antibody (H-414; Santa Cruz Biotechnology) and DyLight
594-conjugated secondary antibody (Jackson ImmunoResearch). Flow cytometry analysis was
performed on an LSRII FACS or a FACSAria cell sorter (BD Biosciences), and analyzed using
FlowJo software (FlowJo).

BM cells were harvested from long leg bones and resuspended in staining medium consisting of biotin-, flavin and phenol red-deficient RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 2% FBS (Hyclone, Logan, UT), 10 mM HEPES (pH 7.2), 1 mM EDTA and 0.02% sodium azide. After filtering through 70 μm nylon mesh, BM cells were incubated with anti-CD16/32 antibody (BioXCell, West Lebanon, NH) for 10 min on ice to block Fc receptors, and then incubated with primary antibodies for 20 min. The lineage cocktail contained biotin-conjugated antibodies to B220 (RA3-6B2), CD3e (145-2C11), CD11b (M1/70), CD19 (1D3), Ly-6G (RB6-8C5) and Ter-119 (TER-119). Additional antibodies for HSC analysis were Ly-6A/E(Sca1)-FITC or -PerCp-Cy5.5 (D7), CD117(c-Kit)-PE-Cy7 or -APC (2B8), CD135(Flt3)-PE (A2F10) and CD150-APC (mShad150). All antibodies were purchased from eBioscience (San Diego, CA). Cells stained with biotin-labeled antibodies were incubated with streptavidin-eFluor 450 (eBioscience) for 15 min on ice and washed three times with staining medium. After the final wash, cells were resuspended in staining medium with 1 μg/ml propidium iodide to exclude dead cells. Flow cytometry analysis was performed on a 5-laser, 18-detector LSR II FACS (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (Treestar, Ashland, OR).