Purification of A2B5+ cells was carried out as described previously (Liu et al., 2004 (link)). Briefly, upon completion of directed differentiation of iPSCs, cells were harvested using Accutase and resuspended in 1% fetal bovine serum (FBS) in 1× phosphate buffered saline (PBS) at a concentration of 5– 10 × 106 cells/mL. Cells were then stained with anti-A2B5 antibody, followed by a FITC-conjugated secondary antibody, and purified using a FACSAria II cell sorter system (BD) at 4 °C, at a rate of 2500 cells/s. The sorted cells were re-examined by FACS for the percentage of A2B5+ and TRA1-81+ cells to determine purity. All experiments were done in triplicate.
Facsaria 2 cell sorter system
Manufactured by BD
The FACSAria II cell sorter system is a high-performance flow cytometry instrument designed for cell analysis and sorting. It is capable of simultaneously detecting and separating multiple cell populations based on their physical and fluorescent characteristics.
Automatically generated - may contain errors
Lab products found in correlation
A1933, by Merck Group (1 mentions)
Diva program, by BD (1 mentions)
Fluorescein isothiocyanate conjugated fitc anti human annexin 5 apoptosis detection kit 1, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Rlt plus lysis buffer, by Qiagen (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
5 protocols using facsaria 2 cell sorter system
1
Purification of A2B5+ Cells from iPSCs
2
Purification of A2B5+ Cells from iPSCs
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Purification of A2B5+ cells was carried out as described previously (Liu et al., 2004 (link)). Briefly, upon completion of directed differentiation of iPSCs, cells were harvested using Accutase and resuspended in 1% fetal bovine serum (FBS) in 1× phosphate buffered saline (PBS) at a concentration of 5– 10 × 106 cells/mL. Cells were then stained with anti-A2B5 antibody, followed by a FITC-conjugated secondary antibody, and purified using a FACSAria II cell sorter system (BD) at 4 °C, at a rate of 2500 cells/s. The sorted cells were re-examined by FACS for the percentage of A2B5+ and TRA1-81+ cells to determine purity. All experiments were done in triplicate.
3
Sorting GD2 and TIE2 Positive Cells
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The NP-like cells after differentiation were digested to single cells using Accutase after washing with PBS, and then stained with GD2 and TIE2 antibodies followed by incubation with fluorescently labeled secondary antibodies. Finally, cells were re-suspended in cool PBS containing 0.5% BSA (A1933, Sigma) at a concentration of 2x 10 6 /ml. Appropriate isotype controls were used. Cells were sorted using a FACSAria II cell sorter system (BD) to determine the percentage of cells positive for GD2 and TIE2 (% cells).
4
Apoptosis Detection with Flow Cytometry
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Propidium iodide (BD Bioscience) and the fluorescein isothiocyanate-conjugated (FITC) anti-human Annexin V Apoptosis Detection Kit I (BD Pharmingen) were used to characterize cells. Labeled cells (1 × 106) were analyzed using the BD FACS Aria II Cell Sorter System (BD Biosciences), followed by data analysis using the Diva program (BD Biosciences).
5
Immune Cell Phenotyping by FACS
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B16-F10 cells or activated WT T-cells were harvested, washed, and incubated with FVDeF780, TruStain FcX PLUS, rPD-L1, anti-PD-1, anti-CD-3, or respective control abs, as above. 29F.1A12+/−, RMP1-30+/−, or rPD-L1+/− cohorts negative for FVDeF780 were directly sorted into RLT Plus lysis buffer (Qiagen) supplemented with β-mercaptoethanol (Sigma) using a BD FACS Aria™ II cell sorter system, for subsequent qRT-PCR analyses as described above.
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