1 × 105 MCF7 cells were treated with atovaquone (5 μM and 10 μM) for 48 hours in 6-well plates, grown as a monolayer. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 12 hours, MCF7 cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen cat.559942) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Invitrogen) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.
Iotest cd24 pe
Manufactured by Beckman Coulter
The IOTest CD24-PE is a reagent used for the identification and enumeration of CD24-positive cells by flow cytometry. It contains an anti-CD24 antibody conjugated with the fluorescent dye phycoerythrin (PE). The reagent can be used to study the expression of CD24, a cell surface glycoprotein, in various cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Apc mouse anti human cd44, by BD (6 mentions)
Fixable dead violet reactive dye, by Thermo Fisher Scientific (4 mentions)
Live dead dye, by Thermo Fisher Scientific (4 mentions)
Fortessa, by BD (4 mentions)
Fluorescence activated cell sorting (facs), by BD (2 mentions)
Apc mouse anti human cd44 559942, by BD (1 mentions)
Dapi dye, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
7 aminoactinomycin d, by Thermo Fisher Scientific (1 mentions)
7 protocols using iotest cd24 pe
1
Atovaquone Modulates Breast Cancer Stemness
2
Quantifying CSC Subpopulation in MCF7 Cells
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Four hundred thousand MCF7 cells were seeded either in either a regular 10 cm dish or in five 15 cm dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA, P3932, Sigma) in the presence of mDIVI1 or vehicle for 2 days or 5 days. Following mDIVI1 treatment, MCF7 cells grown either in suspension or as monolayers were analysed for their expression of CD24 and CD44. The surviving fraction after 2 days and 5 days of growth was analyzed by FACS. MCF7 cells were either trypsinised or spun down and trypsinised and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse anti-human CD44, 559942, BD Pharmingen) for 15 minutes on ice. Cells were then rinsed twice in PBS, spun down, and resuspended with DAPI dye (D1306, Molecular probes) at 3 µM in PBS for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live cell population, identified using the DAPI staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software. Only those cells expressing CD44 that did not express CD24 were considered to be the CD24−/CD44+ CSC subpopulation.
3
Quantifying CD24/CD44 Expression in MCF7 Cells
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1 × 105 MCF7 cells were plated in 6-well plates in complete media supplemented with 10% heat-inactivated FBS. Next day, cells were treated with DPI (5, 10, 50 nM) for 5 days. Vehicle alone (DMSO) control cells were processed in parallel. Briefly, 30,000-50,000 live cells, as identified by 7-AAD dye staining, were analyzed for CD24/CD44 expression. We employed CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen) antibodies for FACS-analysis, using the BD LSR Fortessa (BD Bioscience). Results are the average of three biological replicates (repeats) and are expressed as percentages of mean fluorescence intensity, normalized to the control. * p<0.05, ** p<0.01, *** p<0.001. One-way ANOVA was used with Bonferroni's multiple comparisons test.
4
Enrichment and Analysis of Cancer Stem Cells
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MCF7 and MCF7 DoxyR cells were seeded on low-attachment plates to enrich for the CSC population [54 (link)]. Under these conditions, the non-CSC population undergoes anoikis (a form of apoptosis induced by a lack of cell-substrate attachment) and CSCs are believed to survive. The surviving CSC fraction was analyzed by FACS analysis. Briefly, 1 × 105 MCF7 and MCF7 DoxyR monolayer cells were seeded for 48h in 6-well plates. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 10h, cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Life Technologies) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.
5
Enrichment of Breast Cancer Stem Cells
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Following GO treatment, the CSC population was enriched by seeding on low-attachment plates. Under these conditions, the non-CSCs undergo anoikis (a form of apoptosis induced by lack of proper attachment) and CSCs are believed to survive. The expression of differentiation markers by the surviving “CSC fraction” was analyzed by FACS analysis. Briefly, 1 × 104 MCF7 cells were treated with GO (50μg/ml) for 48h in 6-well plates, grown as a monolayer. Then, the monolayer cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 10h under low-attachment conditions, MCF7 cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen cat.559942) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Invitrogen) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analysed using FlowJo software. Virtually identical results were also obtained using 7-AAD (7-Aminoactinomycin D; Life Technologies) to distinguish between the live and dead populations of cells (cell viability), during anoikis.
6
Enrichment and Analysis of TICs
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Following XCT790 treatment for 48 hours, the TIC population was enriched by seeding on low-attachment plates. Under these conditions, the non-TIC population undergoes anoikis (a form of apoptosis induced by a lack of cell-substrate attachment) and TICs are believed to survive. The surviving TIC fraction was analyzed by FACS analysis. Briefly, 1 × 104 MCF7 monolayer cells were treated with XCT790 (5μM and 10 μM) for 48h in 6-well plates. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 10h, MCF7 cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen cat.559942) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Invitrogen) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.
7
Bedaquiline Modulates Breast Cancer Stemness
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1 × 105 MCF7 cells were treated with bedaquiline (5 μM and 10 μM) for 48 hours in 6-well plates, grown as a monolayer. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 12 hours, MCF7 cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen cat.559942) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Invitrogen) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.
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