H 2kd

Spleen cells were first incubated with anti-murine Fcγ receptor II/III mAb, 2.4G2 for 10 min and then stained with saturating concentrations of Alexa Fluor 488-conjugated, APC-conjugated, biotin-conjugated, PE-conjugated, FITC-conjugated, PerCPCy5.5-conjugated, Alexa Fluor 647-conjugated and Pacific Blue-conjugated mAb against CD3, CD4, CD8, CD19, B220, H2-K^b, I-A^b, H-2K^d, and I-A^d, purchased from either BD Biosciences (San Jose, CA), BioLegend (San Diego, CA), eBioscience (San Diego, CA), or Invitrogen (Carlsbad, CA). Biotinylated primary mAb were detected using PE-Texas Red-streptavidin (BD Biosciences, San Jose, CA). Cells were fixed in 1% paraformaldehyde before reading.
Multi-color flow cytometric analyses were performed using a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Gating strategies: lymphocytes were gated by forward and side scatter and fluorescence data were collected for a minimum of 10,000 gated cells. Studies of donor T cells were performed on a minimum of 5,000 cells collected using a lymphocyte gate that was positive for CD4 or CD8 and either positive (host origin) or negative (donor origin) for MHC class I of the uninjected parent e.g. for B6→F1 mice donor cells are H-2K^d negative. B cells were gated as positive for B220 and either positive (host origin) or negative (donor origin) for MHC Class II of the uninjected parent.

The animal studies were conducted following the protocols approved by the University Committee on the Use and Care of Animals at the University of Michigan. Female NOD/SCID mice (Jackson Laboratory, Bar Harbor, ME, USA) were used to evaluate the effects of LRP8 knockdown on tumor growth and tumorigenicity. Briefly, 5000 SUM149 cells carrying doxycycline-inducible control or LRP8 shRNA were injected into the inguinal mammary fat pad of 6–8-week-old mice. Doxycycline diet (625 mg/kg) (Envigo, Haslett, MI, USA) was given to mice starting from 5 weeks after implantation when palpable tumors were observed. At the end of tumor growth monitoring, tumors were harvested and dissociated by using Tumor Dissociation Kit, human (Miltenyi Biotec, Auburn, CA, USA), and then DAPI and H-2Kd (BD Biosciences) double-negative live human cancer cells were sorted by flow cytometry. Limiting dilution assay was conducted by inoculating the sorted and serially diluted cancer cells (2,500, 500 and 100 cells/inoculation) into the inguinal mammary fat pad of tumor-free mice. Tumor formation was monitored for 3 months. The frequency of tumor-initiating cells was calculated by using Extreme Limiting Dilution Analysis (ELDA) [25 ].