Anti cd8a ly 2

A detailed protocol of this procedure was submitted to protocol exhange⁶⁷ .
Bone marrow cells from adult BALB/cJ male or female mice, ages 8-12 weeks, were lineage-depleted using the Mouse Streptavidin RapidSpheres Isolation Kit (STEMCELL Technologies [Cat# 19860A]), with the following biotinylated antibodies:

anti-CD11b (Clone M1/70 [#557395], BD Biosciences)

anti-Ly-6G and Ly-6C (Clone RB6-8C5 [#553125], BD Biosciences)

anti-CD4 (Clone RM4-5 [#553045], BD Biosciences)

anti-CD8a (Ly-2) (Clone 53-6.7 [#553029], BD Bioscience)

anti-CD19 (Clone 1D3 [#553784], BD Biosciences)

anti-TER119 (Clone TER119 [#553672], BD Biosciences).

Lineage-depleted cells were then labeled with the following antibodies in the presence of 1% rat serum:

streptavidin Alexa Fluor 488 (Molecular Probes), to mark lineage-positive cells

CD117-APC Cy7 (Clone 2B8 [#105826], Biolegend)

TER119-BUV395 (Clone TER-119 [#563827], BD Biosciences)

CD71-PE Cy7 (Clone RI7217 [#113812], Biolegend)

CD55-AF647 (Clone RIKO-3 [#131806], Biolegend)

CD105-PE (Clone MJ7/18 [#120408], Biolegend)

CD150-BV650 (Clone TC15-12F12.2 [#115931], Biolgened)

CD41-BV605 (Clone MWReg30 [#133921], Biolegend)

CD49f (=itga6) – BV421 (Clone GoH3 [#313624], Biolegend)

Following washes, cells were re-suspended in DAPI-containing buffer and sorting was performed on BD FACSAria II with a 100 μ nozzle. Sorted populations were defined as in Fig. 2a.

Femurs and tibiae were harvested immediately following euthanasia, and placed in cold (4°C) ‘staining buffer’ (phosphate-buffered saline (PBS) containing 0.2% Bovine Serum Albumin (BSA) and 0.08% Glucose). Bones were flushed using a 2 ml syringe with a 26-gauge needle and then crushed with a pestle and mortar to obtain any remaining cells. Flushing the bones is gentler than crushing, promoting cell viability; however, cell yield is lower, and some cell types may be protected from flushing in bone niches that are less accessible to flushing. For this reason, we first flush the bones, obtaining as many cells as possible, and then crush the bones to obtain any remaining cells. Harvested bone marrow cells were filtered through a 70 µm strainer and washed in cold ‘Easy Sep’ buffer (PBS; 2% fetal bovine serum (FBS); 1mM EDTA). Harvested BM cells were lineage-depleted using the Mouse Streptavidin RapidSpheres Isolation Kit (STEMCELL Technologies [Cat# 19860A]), with the following biotinylated antibodies:

anti-CD11b (Clone M1/70 [#557395], BD Biosciences)

anti-Ly-6G and Ly-6C (Clone RB6-8C5 [#553125], BD Biosciences)

anti-CD4 (Clone RM4-5 [#553045], BD Biosciences)

anti-CD8a (Ly-2) (Clone 53-6.7 [#553029], BD Bioscience)

anti-CD19 (Clone 1D3 [#553784], BD Biosciences)

anti-TER119 (Clone TER119 [#553672], BD Biosciences).