The cells were grown to confluency, washed two times with phosphate buffered saline (PBS) and detached using Accutase (BD Biosciences, San Jose, CA). The cell pellet was washed and re-suspended in BD Pharmingen™ stain buffer (BSA). The cells were counted and adjusted to a concentration of 1×106 cells/mL. For every 100 uL of cell suspension, 10 uL of FITC conjugated CD133 (Miltenyi Biotec) and/or PE conjugated CD24 antibody (Miltenyi Biotec) was added to the tubes. The cells were incubated for 30 min on ice in the dark and then washed two times with stain buffer and re-suspended in 700 ul of stain buffer. Cells were counted with a BD LSRII and the data was analyzed using the FlowJo software. For each sample, 10,000 events were acquired. Results were gated to exclude doublets and identify the singlet population.
Fitc conjugated cd133
Manufactured by Miltenyi Biotec
Sourced in United States
FITC conjugated CD133 is a fluorescent-labeled antibody targeting the CD133 antigen. CD133 is a cell surface glycoprotein that is expressed on various stem and progenitor cell populations. The FITC conjugate allows for the detection and analysis of CD133-positive cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Accutase, by BD (2 mentions)
Lsr 2, by BD (1 mentions)
Pharmingen stain buffer, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Pe conjugated cd44, by BD (1 mentions)
Pharmingen stain buffer bsa, by BD (1 mentions)
C6 flow cytometer, by BD (1 mentions)
Dynamag 2 magnet, by Thermo Fisher Scientific (1 mentions)
4 protocols using fitc conjugated cd133
1
Immunophenotyping of Stem Cell Markers
2
Quantifying Autophagy and Stem Cell Markers
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1 × 105 cells of each sample was incubated for 15 minutes in 1:1000 dilution of 1 µg/ml acridine orange and autophagy was quantified following71 (link). For CD133 and CD44 expression analysis, 1 × 105cells were blocked for 1 hour in 0.3% NGS for 1 h followed by incubation in manufacturer specified dilution primary antibody (PE conjugated CD44 (BD, USA) and FITC conjugated CD133 (Miltenyi, USA)) for 1 hour. The cells were washed and the expression was measured in BD FACS CANTOII flow cytometer (BD, USA). Viable cells were gated and the expression of CD44 and CD133 were measured post compensation. The analysis was performed using FLowJo software (Flowjo USA).
3
Identification of RCC Stem Cell Subpopulations
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Confluent cultures of the renal cell carcinoma (RCC) cell lines (HTB44, HTB47 and CRL1933) were washed two times with PBS and the cells were detached using Accutase (BD Biosciences) and centrifuged at 2000 rpm at 4 °C for 5 min. The cell pellet was washed once and re-suspended in BD Pharmingen™ stain buffer (BSA). Cells were diluted to 1 × 106 cells/mL with the stain buffer. For every 100 µL of cell suspension, 10 µL of fluorescein isothiocyanate (FITC) conjugated CD133 and/or phycoerythrin (PE) conjugated CD24 antibody (Miltenyi Biotec Inc., Gaithersburg, MD, USA) was added to the tubes. The cells were incubated for 30 min on ice in the dark and then washed two times with stain buffer and re-suspended in 1 mL of stain buffer. Cells were analyzed using SONY flow cytometer SP800S. The singlet population was gated as FSC vs. SSC, following which the selected population was graphed as PROM1 vs. CD24 plot. Four-quadrant data analysis was used to separate double positive vs. single positive cell populations. Based on the compensation measurements, the threshold for positive signal was set to ≤1 × 103 for both markers at the x and y-axis.
4
Isolation and Characterization of Glioblastoma Stem Cells
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CD133 has been used to enrich the putative cancer stem cells [25 (link), 26 (link)]. In this study, the anti-CD133-conjugated microbeads were applied to isolate GSCs from U-251 cells by using magnetic activated cell sorting (MACS) as previously reported with slight modification [40 (link)]. In brief, U-251 MG cells were incubated with anti-CD133-conjugated microbeads antibody (10 μl anti-CD133 microbeads per 107 U251 cells) in 100 μl reaction volume for 20 mins in the refrigerator. Then, the CD133+ cells were collected by using a magnet separator (DynaMag-2 magnet; Thermo scientific). The purity of GSCs was confirmed by flow cytometry analysis. The purified GSCs were expanded in DMEM/F12 medium containing 2% B27 (without retinoic acid), EGF (20 ng/ml), FGF-2 (20 ng/ml), heparin (5 μg/ml), glutamine (2 mM) and 1% antibiotics.
For flow cytometry analysis, the purified GSCs and U-251 MG cells were washed with PBS twice, and then incubated with FITC-conjugated CD133 (5 μl/1×106 cells, Miltenyi Biotec), or isotype control antibody (FITC-conjugated IgG, 20 μl/1×106 cells, BD biosciences) for 30 mins in the dark. After incubation, the samples were analyzed under flow cytometry (BD C6 flow cytometer). 10,000 events were collected for analysis. The experiment was repeated three times.
For flow cytometry analysis, the purified GSCs and U-251 MG cells were washed with PBS twice, and then incubated with FITC-conjugated CD133 (5 μl/1×106 cells, Miltenyi Biotec), or isotype control antibody (FITC-conjugated IgG, 20 μl/1×106 cells, BD biosciences) for 30 mins in the dark. After incubation, the samples were analyzed under flow cytometry (BD C6 flow cytometer). 10,000 events were collected for analysis. The experiment was repeated three times.
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