FACS was utilized to sort breast CSCs according to cluster of differentiation 44 (CD44) and cluster of differentiation 24 (CD24) cell surface markers. Briefly, MDA-MB-231 cells were stained with FITC-labeled mouse anti-human CD44 antibodies and phycoerythrin (PE)-labeled mouse anti-human CD24 (BD Pharmingen™) antibodies. Unbound antibodies were removed by washing with phosphate-buffered saline (PBS) prior to analysis. Then, CD44+/CD24− cells and non-CD44+/CD24− cells were collected.
Mouse anti human cd44
Manufactured by BD
Sourced in United States
Mouse anti-human CD44 is a primary antibody that recognizes the CD44 antigen, a cell surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. It is commonly used in research applications to identify and study CD44-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti human cd34, by BD (3 mentions)
Mouse anti human cd45, by BD (2 mentions)
Mouse anti human cd105, by BD (2 mentions)
Mouse anti human cd73, by BD (2 mentions)
Pe labeled mouse anti human cd24, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Flow tube, by Corning (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Mouse anti human cd9, by R&D Systems (1 mentions)
Goat anti human nanog, by R&D Systems (1 mentions)
Cd151, by BD (1 mentions)
Mouse anti human hla dr, by BD (1 mentions)
Mouse anti human cd90, by BD (1 mentions)
Mouse anti human p75ntr, by BD (1 mentions)
Cyflow space flow cytometer, by Sysmex (1 mentions)
6 protocols using mouse anti human cd44
1
Breast Cancer Stem Cell Isolation Using FACS
2
Characterizing hiPSC-derived MSCs and rat BMSCs
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The hiPSC-derived MSCs or rat BMSCs were harvested using 0.25% trypsin/ethylenediaminetetraacetic acid and resuspended in 100 μl staining medium (2% FBS and 2% N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid in PBS). The cells were stained with mouse anti-human CD29 (BD, USA), mouse anti-human CD44 (BD, USA), mouse anti-human CD34 (BD, USA), mouse anti-human CD105 (BD, USA), and mouse anti-human CD45 (BD, USA) antibodies for 30 min at 4℃. Isotype-matched antibody (immuno-globulin G 2b-fluorescein isothiocyanate) was used to determine nonspecific fluorescence. Samples were run on a cytometer (CytoFLEX Beckman Coulter, USA).
3
Isolation and Characterization of Urine-Derived Stem Cells
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Primary culture of the USCs was performed as previously described [33 (link),34 (link)]. Briefly, the USCs were obtained from four young male adult donors (20–30 years old). Fresh urine sample (about 200 mL) was centrifuged and washed with PBS for twice. The sediment was re-suspended and cultured in T25 cell culture flasks with 5 mL of established medium [35 (link)].
Cells in logarithmic growth phase were digested with trypsin (Gibco, USA), with their density adjusted to 1 × 106 cells/mL with PBS. 100 μL aliquot of cell suspension was moved to each flow tube (Corning, USA). Thereafter, 2 μL of mouse anti-human CD29 (559883, BD Pharmingen™, USA), mouse anti-human CD34 (555821, BD Pharmingen™), mouse anti-human CD44 (555478, BD Pharmingen™), mouse anti-human CD73 (550257, BD Pharmingen™), mouse anti-human CD90 (561558, BD Pharmingen™) and mouse anti-human CD133(130-111-085, Miltenyi Biotec GmbH, Germany) antibodies were added to each tube. The mixture was allowed to incubate for 30 min in darkness at room temperature. Thereafter, the cells were washed twice with PBS and re-suspended in 200 μL PBS for flow analysis.
Cells in logarithmic growth phase were digested with trypsin (Gibco, USA), with their density adjusted to 1 × 106 cells/mL with PBS. 100 μL aliquot of cell suspension was moved to each flow tube (Corning, USA). Thereafter, 2 μL of mouse anti-human CD29 (559883, BD Pharmingen™, USA), mouse anti-human CD34 (555821, BD Pharmingen™), mouse anti-human CD44 (555478, BD Pharmingen™), mouse anti-human CD73 (550257, BD Pharmingen™), mouse anti-human CD90 (561558, BD Pharmingen™) and mouse anti-human CD133(130-111-085, Miltenyi Biotec GmbH, Germany) antibodies were added to each tube. The mixture was allowed to incubate for 30 min in darkness at room temperature. Thereafter, the cells were washed twice with PBS and re-suspended in 200 μL PBS for flow analysis.
4
Characterization of Pluripotent Stem Cells
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Cells were suspended and washed three times with washing buffer composed of ice-cold D-PBS, 1% bovine serum albumin (BSA), 5 mM EDTA, and 25 mM Hepes before antibody incubation. hiPSCs were incubated with the primary antibody goat anti-human OCT3/4 (R&D Systems, Minneapolis, MN, USA), mouse anti-human SOX2 (R&D Systems), goat anti-human NANOG (R&D Systems), mouse anti-human PODPCALYXIN (R&D Systems), mouse anti-human SSEA4 (R&D Systems), or mouse anti-human CD9 (R&D Systems) for 30 min at 4°C. After three subsequent washes with the buffer, cells were incubated with secondary antibody Alexa Fluor 546 donkey anti-mouse (Thermo Fisher Scientific) or fluorescein isothiocyanate (FITC) donkey anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA) for another 30 min at 4°C. For analysis of MC-Chs and NCC-Chs, cells were incubated with phycoerythrin-conjugated mouse anti-human CD44 or CD151 (BD) for 30 min at 4°C. Fluorescent cells were then detected by the Attune NxT Flow Cytometer (Life Technologies, Carlsbad, CA), and the resulting flow cytometry data were analyzed by FlowJo (Tree Star, Ashland, OR), following the manufacturer’s instructions.
5
Multiparametric Flow Cytometry Analysis of Stem Cell Markers
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Fourth‐passage cells (5 × 105) of each group were harvested and fixed with 4% paraformaldehyde for 30 minutes, followed by incubation with mouse anti‐human p75NTR, mouse anti‐human CD44, mouse anti‐human CD73, mouse anti‐human CD90, mouse anti‐human CD105, mouse anti‐human CD11b, mouse anti‐human CD19, mouse anti‐human CD34, mouse anti‐human CD45 and mouse anti‐human HLA‐DR antibodies (1:20; BD Pharmingen™) at 4°C for 2 hours. Subsequently, the specimens were analysed by flow cytometry.
6
Breast Cancer Stem Cell Phenotyping
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For the analysis of the expression of cell surface breast CSC markers, cells were incubated with 10 μL of APC Mouse Anti-Human CD44 (BD Pharmingen™, cat. no. 559942, Franklin Lakes, NJ, USA), PE Mouse Anti-Human CD24 (BD Pharmingen™, cat. no. 555428, Franklin Lakes, NJ, USA) as with APC and PE isotype control antibodies for 20 min at RT in the dark. Stained cells were analyzed on a flow cytometer CyFlow space (Sysmex PARTEC GmbH, Kobe, Japan). Data analysis was performed with FloMax software (Partec) and representative plots were performed with FlowJo software (LLC).
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