MDA-MB-231-luc-D3H1 cells were stained with the following antibodies: FITC Rat Anti-human CD49f, PE Mouse Anti-human CD44, Alexa fluor 647 Mouse Anti-human CD24 (BD Pharmingen™). Fluorescence-activated cell sorting BD, FACSAria II SORP, and FACSDiva 8.0.1 software were used. Stained cells were examined and sorted for stem/progenitor cells with CD49f+ population followed by a final gate to select CD44+/CD24- within the CD49+ cell fraction. A total of 1.39x106 cells were sorted. The percentage of parent population were 98.31 of CD49f+ marker, followed by a sequential gate of CD24+ and CD44+ with 0.01% and 1.30% of parental population respectively. After sorting, cells were seeded in ultra-low adherent plates for tumorsphere formation assays and treated with SFN or control (0.9% NaCl) for 7–10 days.
Rat anti human cd49f
Manufactured by BD
Rat anti-human CD49f is a laboratory reagent used for the detection and identification of human CD49f, a cell surface protein. It can be used in various immunological techniques, such as flow cytometry and immunohistochemistry, to analyze the expression of CD49f on cells.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti human cd34, by BD (2 mentions)
Mouse anti human cd90, by BD (2 mentions)
Mouse anti human cd38, by BD (2 mentions)
Mouse anti human cd45ra, by BD (2 mentions)
Facsaria 2 sorp, by BD (1 mentions)
Pe mouse anti human cd44, by BD (1 mentions)
Alexa fluor 647 mouse anti human cd24, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Ab131260, by Abcam (1 mentions)
Lsrfortessa, by BD (1 mentions)
Ab150077, by Abcam (1 mentions)
Intrasuretm kit, by BD (1 mentions)
3 protocols using rat anti human cd49f
1
Isolation and Culture of Breast Cancer Stem Cells
2
Multiparametric Flow Cytometry Analysis of Immune Cell Populations
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Cells were washed with ice-cold phosphate-buffered saline (PBS) and stained with corresponding antibodies in PBS containing 2% fetal bovine serum and 0.02% sodium azide. We used: mouse anti-human CD38 (BD, #563964), mouse anti-human CD34 (BD, #348811), rat anti-human CD49f (BD, #563271), mouse anti-human CD90 (BD, #562685), and mouse anti-human CD45RA (BD, #560673). For intracellular NE protein analysis, cells were subsequently fixed and permeabilized using the Fix&Perm kit (Nordic Mubio, #GAS-002FOC), followed by incubation with a rabbit anti-human NE (abcam, #ab131260) antibody for 15 minutes (min) at room temperature, and subsequent incubation with a goat polyclonal secondary antibody to rabbit IgG-H&L (Alexa Fluor 488) (abcam, #ab150077) for 15 min at room temperature. Cells were fixed with 0.5% paraformaldehyde and measured using a BD LSRFortessa.
Additional methods are available in the Online Supplementary Appendix.
Additional methods are available in the Online Supplementary Appendix.
3
Characterizing Hematopoietic Stem Cells
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BM mononuclear cells were washed with phosphate-buffered saline (PBS) and stained with corresponding surface marker antibodies in PBS containing 2% fetal bovine serum (FBS) and 0.02% sodium azide. For surface marker analysis (panel adapted from van Galen et al. 21 ), the following antibodies were used: mouse anti-human CD38 (BD, #563964), mouse anti-human CD34 (BD, #348811), rat anti-human CD49f (BD, #563271), mouse antihuman CD90 (BD, #562685), mouse anti-human CD45RA (BD, #560673), mouse anti-human CD10 (BD, #563734), and mouse anti-human CD135 (BD, #564708). For the analysis of intracellular gH2AX protein, cells were subsequently permeabilized and fixed using the IntraSure TM Kit (BD #641776), followed by incubation with mouse anti-human gamma gH2AX (BD, #560445) antibody. For the analysis of ROS, cells were incubated with 20 μM 2ʹ,7ʹ-dichlorofluorescin diacetate (DCFH-DA) (Sigma-Aldrich/Merck, #D6883) for 10 min at 37 °C after staining with surface marker antibodies.
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