Anti cd31 biotin

The CD133⁺ population was separated from the mouse progenitor cells and other CD133⁻ cells using MACS separation (Miltenyi Biotech) using manufacturers protocol. Single cell suspension was generated from tumors in KPC mice according to the Li et al [44 ]. Non-epithelial progenitor cells were removed using anti-CD31-Biotin (BD Biosciences) and anti-CD45 Biotin (BD Bioscience) using MACS technique. The flowthrough free from the mouse progenitor cells was bound to anti-mouse CD133⁻Microbeads for 10 min on ice and positively purified for CD133⁺ cells by MACS. The purity of separation was tested for each batch by performing a FACS analysis using Anti-CD133⁻PE antibody AC141 (Miltenyi Biotech). The separated populations were used for RNA, Protein and FACS analysis. Cells growing in culture were scraped gently into centrifuge tube and washed once in Wash Buffer (PBS, 0.5% BSA, 2 mM EDTA) before binding to Anti-mouse CD133 microbeads and proceeding as described above.
For labeling with ¹³C glucose, isolated CD133⁺ cells were incubated in glucose free DMEM for 30 min. Following incubation, the cells were transferred to a growth medium (DMEM) with ¹³C glucose (final concentration 25 mM) and incubated further for 30 min. Reaction was quenched and stored in −20°C until further use. CD133⁻ cells were processed in parallel with ¹²C glucose (final concentration 25 mM) added to DMEM.

The CD133⁺ population was separated from the mouse progenitor cells and other CD133⁻ cells using MACS separation (Miltenyi Biotech) following manufacturers protocol. Single cell suspension was generated from tumors in KPC mice according to Li et al.^{25 (link)}. Non-epithelial progenitor cells were removed using anti-CD31-Biotin (BD Biosciences) and anti-CD45 Biotin (BD Bioscience) using MACS technique. The flowthrough free from the mouse progenitor cells was bound to anti-mouse CD133⁻ Microbeads for 10 min on ice and positively purified for CD133⁺ cells by MACS. The purity of separation was tested for each batch by performing a FACS analysis using Anti-CD133-PE antibody AC141 (Miltenyi Biotech).