Suspensions of BM cells were enriched for Gr-1dimCD11b+ and Gr-1hiCD11b+ populations using two-step magnetic cell sorting. Briefly, BM cells were stained with PE-anti-Gr-1 mAbs (eBioscience, San Diego, CA), incubated with anti-PE MultiSortMicroBeads (MiltenyiBiotec, San Diego, CA), washed and separated by sequential passage through LS MACS column (Miltenyi Biotec). The positive fraction was enriched for Gr-1hi cells (purity, 60–75%, all CD11b+, Fig. 2F ). The negative fraction was composed of Gr-1dimCD11b+ and Gr-1negCD11b− cells. For the isolation of Gr-1dimCD11b+ cells, the negative fraction was incubated with APC-anti-CD11b mAbs, magnetically labeled with anti-APC MultiSort MicroBeads (Miltenyi Biotec), and sorted on LS MACS column to collect the positive fraction. The later contained mostly Gr-1dimCD11b+ cells (purity, 90–95%, Fig. 3A ).
Anti pe multisortmicrobeads
Manufactured by Miltenyi Biotec
Anti-PE MultiSortMicroBeads are magnetic beads coated with an anti-PE (Phycoerythrin) antibody. They are designed for the magnetic separation and purification of PE-labeled cells or target molecules from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Macs ls column, by Miltenyi Biotec (1 mentions)
Anti apc multisort microbeads, by Miltenyi Biotec (1 mentions)
Flow cytometry, by BD (1 mentions)
Cd133 pe antibody, by Miltenyi Biotec (1 mentions)
Cd44 microbeads, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Cd34 microbead kit, by Miltenyi Biotec (1 mentions)
3 protocols using anti pe multisortmicrobeads
1
Enrichment of Myeloid Subsets from Bone Marrow
2
Isolation of Cancer Stem Cell Subpopulations
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Cells positive for CD44 and CD133 surface markers were isolated from cultured Hep2 and TU-177 cells by MACS by using CD44 MicroBeads or CD133-PE antibody (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's instructions. Briefly, 107 cells in 100 μL buffer were incubated with 10 μL CD133-PE antibody. Cells were washed with 1 mL buffer and centrifuged at 300×g for 10 min, then the cell pellet was resuspended with 90 μL buffer, and 10 μL anti-PE Multisort MicroBeads (Miltenyi Biotec) was added and incubated for 15 min in the dark at 4℃. Cells were washed and underwent magnetic sorting, then CD133-enriched (CD133+) and CD133-depleted (CD133-) cell populations were collected. For 107 cells of CD133+ or CD133-, 20 μL CD44 MicroBeads was added and incubated for 15 min in the dark at 4 ℃, followed by washing and magnetic sorting, then CD133+CD44+, CD133+CD44-, CD133-CD44+, and CD133-CD44- cells were obtained. After isolation, cells with the 4 types of surface markers were cultured for further analysis, and some of these cells were used to evaluate the efficiency of magnetic separation by flow cytometry (Becton Dickinson).
3
Isolation and Characterization of Cord Blood Immune Cells
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All CB samples were obtained with prior written consent and ethical committee approval from the Anthony Nolan Cord Blood bank (Research Ethics Committee reference 10/H0405/27). The study had full ethical approval from the Anthony Nolan and Royal Free Hospital Research Ethics Committee. CB mononuclear cells (CBMCs) were isolated by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). CBSC were isolated using the CD34 microbead kit (Miltenyi Biotec) [30 ] to a purity of 98.4% ± 0.75. CBSC purity was analyzed as CD133+CD34+CD45low and following the International Society of Hematotherapy and Graft Engineering (ISHAGE) gating guidelines. CB NK cells were isolated using the NK cell isolation kit (Miltenyi Biotec), to a purity of 90.39% ± 3.35. When indicated, NK cells were activated for 4 h using 20 ng/mL IL-15 and CD69 expression was assessed on NK cells as a measure of activation. T cells were labeled with PE-conjugated CD4 or CD8 antibodies respectively and isolated from CB using anti-PE MultiSort MicroBeads (Miltenyi Biotec) with purities of 90.16% ± 0.76 and 81.66% ± 11.06 respectively. The function of CD4 and CD8 T cells was not analyzed post-isolation.
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