Femur explants were dissected from 5-week old GAS6+/+ or GAS6 −/− mice. Femur dissection was completed as previously described (23 (link)). Sorted G0/G1 FUCCI cells were injected into the marrow space of the femur explants and cultured for 24 hours in α-MEM (Life Technologies, Carlsbad, CA) supplemented with 5% FBS. 24 hours after dissection, media was changed to α-MEM supplemented with 10% FBS and 2.5μM NE or vehicle control. After 48h of culture in experimental conditions, the bone marrows were flushed from the femur explants with FACS buffer (PBS +2% FBS). Mouse cells were depleted using a MACs Mouse Cell Depletion Kit (Miltenyl Biotec, Cat# 130-021-694) using a MACs automated Pro Cell Separator (Miltenyl Biotec). Remaining cells were labeled with a Live/Dead stain (Zombie Green, BioLegend, Cat# 423111), murine IgG2b b haplotype (mH-2Db) (BioLegend, Cat# 111516, PE/Cy7), mCD45 (BioLegend, Cat# 103112, APC), HLA-A,B,C (Biolegend, Cat# 311426, APC/Cy7). Live cells were negatively gated for mH-2Db and mCD45, and positively gated for HLA-A,B,C prior to cell cycle analysis using the FUCCI spectrum.
Macs mouse cell depletion kit
Manufactured by Miltenyi Biotec
The MACs Mouse Cell Depletion Kit is a laboratory equipment used for the magnetic depletion of unwanted cell populations from mouse cell suspensions. It allows for the isolation of target cells by negatively selecting against specific cell types.
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3 protocols using macs mouse cell depletion kit
1
Murine Femur Explant Culture for FUCCI Cell Analysis
2
Lymphoma Xenograft Model and Biochemical Analysis
3
Isolation of Human Cells from Mouse PDX Tumors
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To separate human and mouse cells from MPT PDX tumours, we used the MACS mouse cell depletion kit (Miltenyi Biotec) designed for isolating human cell populations in xenograft tumours using magnetically labelled beads targeting mouse cells. To dissociate the MPT PDX tumour, we resected the PDX tumour at 1000 mm3 and dissociated it using a tumour dissociation kit with a gentle MACS dissociator (Miltenyi Biotec) according to the manufacturer’s protocol. Dissociated cells were labelled with mouse-specific magnetic beads and separated using LS columns to obtain unlabelled flowing human cells. Following human cell isolation, bead-labelled mouse cells were detached from the LS column.
To verify whether cells were successfully separated by the host species, RNA was extracted from cells collected from each human and mouse using TRIzol reagent. Using a cDNA synthesis kit (Takara), cDNA was synthesised, and qRT-PCR was performed using the human GAPDH primer (F:5’-GAG TCC ACT GGC GTC TTC-3’ R:5’-GGA GGC ATT GCT GAT GAT C-3’) and mouse GAPDH primer (F:5’-GCC TTC CGT GTT CCT ACC-3’ R:5’-GCC TGC TTC ACC ACC TTC-3’).
To verify whether cells were successfully separated by the host species, RNA was extracted from cells collected from each human and mouse using TRIzol reagent. Using a cDNA synthesis kit (Takara), cDNA was synthesised, and qRT-PCR was performed using the human GAPDH primer (F:5’-GAG TCC ACT GGC GTC TTC-3’ R:5’-GGA GGC ATT GCT GAT GAT C-3’) and mouse GAPDH primer (F:5’-GCC TTC CGT GTT CCT ACC-3’ R:5’-GCC TGC TTC ACC ACC TTC-3’).
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