facsariaii high speed cell sorter, by BD - Product details

1

Isolating Erythroblast Subpopulations by Cell Sorting

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To isolate erythroblasts at different stages of maturation by cell sorting, >15 × 10⁶ bone marrow cells from six 7 week-old Sv129/C57BL/6 Fam132b^+/+ littermate mouse femurs (three controls and three 15 hours after phlebotomy) were filtered through a 30 μm filter and resuspended in 1 mL PBS/5% FCS. Cells were blocked with rat anti-mouse CD16/CD32 (0.25 μL/10⁶ cells, BD Biosciences no. 553142) for 15 minutes and subsequently stained with FITC rat anti-mouse TER-119 (0.5 μL/10⁶ cells, BD Biosciences no. 557915), and PE rat anti-mouse CD71 (0.5 μL/10⁶ cells, BD Biosciences no. 553267) and incubated on ice for 30 minutes in the dark. Cells were washed twice with 1 mL PBS and resuspended in 2 mL PBS/5%FCS. Sorting was performed on a FACSAriaII High-Speed Cell Sorter (Becton Dickinson). Erythroblasts population were differentiated as previously described^{17 (link)} into four populations: pro-erythroblasts (pro-E, Ter119^med CD71^high FSC^high), basophilic erythroblasts (Ter119^high CD71^high FSC^high), polychromic erythroblasts (Ter119^high CD71^high FSC^low) and orthochromic erythroblasts (Ter119^high CD71^low FSC^low).

Kautz L., Jung G., Valore E.V., Rivella S., Nemeth E, & Ganz T. (2014). IDENTIFICATION OF ERYTHROFERRONE AS AN ERYTHROID REGULATOR OF IRON METABOLISM. Nature genetics, 46(7), 678-684.

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Isolation and Analysis of Prostate Cancer Stem Cells

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All experimental animal procedures were performed in compliance with the institutional ethical requirements and approved by the University of Michigan Committee for the Use and Care of Animals (UCUCA). PCa^Control or PCa^GAS6OE cells (1 × 10⁶ cells) were injected into male CB.17. SCID mice (4–6 weeks of age, Charles River, Wilmington, MA) by intracardiac (i.c.) injection. Bone marrow cells were flushed from the femorae and tibias 24 hours later and murine hematopoietic lineages were depleted with Miltenyi lineage depletion kit (cat.) using AutoMACS (Miltenyi Boitecs). The enriched cells were incubated with a FITC-anti-HLA-ABC, PE-anti-CD133, and APC-anti-CD44 antibodies. Thereafter, the CD133+/CD44+ and CD133–/CD44– fractions of the HLA positive cells were analyzed with a FACS Aria II High-Speed Cell Sorter (Becton Dickinson, Franklin Lakes, NJ). In some cases, the CD133+/CD44+ and CD133–/CD44– fractions were sorted for gene expression of GAS6 and Mer.

Jung Y., Decker A.M., Wang J., Lee E., Kana L.A., Yumoto K., Cackowski F.C., Rhee J., Carmeliet P., Buttitta L., Morgan T.M, & Taichman R.S. (2016). Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow. Oncotarget, 7(18), 25698-25711.

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Multiparameter Flow Cytometry of Cell Lines

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Cells were stained with FITC- or APC-anti-HLA-ABC antibody (clone W6/32: 311404 (FITC), 311410 (APC), BioLegend, San Diego, CA), PE-cytokeratin antibody (347204, Becton Dickinson, Franklin Lakes, NJ), PE-EpCAM antibody (130-091-253, Miltenyi Biotec), PE-anti-CD133 antibody (130-080-801, Miltenyi Biotec), APC-anti-CD44 antibody (559942, BD Biosciences, San Diego, CA), or isotype-matched IgG control for 20 min at 4°C. Flow cytometric analyses were performed in a FACSAria II High-Speed Cell Sorter (Becton Dickinson).

Shiozawa Y., Berry J.E., Eber M.R., Jung Y., Yumoto K., Cackowski F.C., Yoon H.J., Parsana P., Mehra R., Wang J., McGee S., Lee E., Nagrath S., Pienta K.J, & Taichman R.S. (2016). The marrow niche controls the cancer stem cell phenotype of disseminated prostate cancer. Oncotarget, 7(27), 41217-41232.

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Isolating Erythroblast Subpopulations by Cell Sorting

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To isolate erythroblasts at different stages of maturation by cell sorting, >15 × 10⁶ bone marrow cells from six 7 week-old Sv129/C57BL/6 Fam132b^+/+ littermate mouse femurs (three controls and three 15 hours after phlebotomy) were filtered through a 30 μm filter and resuspended in 1 mL PBS/5% FCS. Cells were blocked with rat anti-mouse CD16/CD32 (0.25 μL/10⁶ cells, BD Biosciences no. 553142) for 15 minutes and subsequently stained with FITC rat anti-mouse TER-119 (0.5 μL/10⁶ cells, BD Biosciences no. 557915), and PE rat anti-mouse CD71 (0.5 μL/10⁶ cells, BD Biosciences no. 553267) and incubated on ice for 30 minutes in the dark. Cells were washed twice with 1 mL PBS and resuspended in 2 mL PBS/5%FCS. Sorting was performed on a FACSAriaII High-Speed Cell Sorter (Becton Dickinson). Erythroblasts population were differentiated as previously described^{17 (link)} into four populations: pro-erythroblasts (pro-E, Ter119^med CD71^high FSC^high), basophilic erythroblasts (Ter119^high CD71^high FSC^high), polychromic erythroblasts (Ter119^high CD71^high FSC^low) and orthochromic erythroblasts (Ter119^high CD71^low FSC^low).

Kautz L., Jung G., Valore E.V., Rivella S., Nemeth E, & Ganz T. (2014). IDENTIFICATION OF ERYTHROFERRONE AS AN ERYTHROID REGULATOR OF IRON METABOLISM. Nature genetics, 46(7), 678-684.

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Facsariaii high speed cell sorter

Lab products found in correlation

4 protocols using facsariaii high speed cell sorter

Isolating Erythroblast Subpopulations by Cell Sorting

Isolation and Analysis of Prostate Cancer Stem Cells

Multiparameter Flow Cytometry of Cell Lines

Isolating Erythroblast Subpopulations by Cell Sorting

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