The isolated blood cells were suspended in staining buffer (2% heat-inactivated fetal calf serum, 0.09% sodium azide in Dulbecco’s PBS; Invitrogen) and blocked using purified rat anti-mice Fc block (BD Pharmingen, San Jose, CA). The viable cell population was analyzed for MSCs with Sca-1-PE(0.2 mg/ml, BioLegend) and CD49a-FITC (0.2 mg/ml, BioLegend) antibody and for EPCs with CD133-PE (0.2 mg/ml, eBioscience) and VEGFR-2-FITC (0.2 mg/ml, Flk-1; BD Parmingen) antibody as previously described respectively [34 (link)–37 (link)]. Isotype-identical antibodies served as controls to determine background staining in every experiment. Flow cytometry was performed using the Beckman Coulter Cytomics FC500 (BD Biosciences, USA).
Cd133 pe
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD133-PE is a cell surface marker conjugated with the fluorescent dye R-Phycoerythrin (PE). It is used for the detection and isolation of CD133-positive cells by flow cytometry and cell sorting.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (2 mentions)
Cd44 fitc, by Thermo Fisher Scientific (2 mentions)
Dulbecco s pbs, by Thermo Fisher Scientific (1 mentions)
Sca 1 pe, by BioLegend (1 mentions)
Cd105 pe, by Thermo Fisher Scientific (1 mentions)
Cd34 pe, by Thermo Fisher Scientific (1 mentions)
Cd31 fitc, by BD (1 mentions)
Cd24 fitc, by BD (1 mentions)
Cd106 fitc, by BD (1 mentions)
Streptavidin apc cy7, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Flt3 pe, by BD (1 mentions)
Cd90.2 fitc, by Thermo Fisher Scientific (1 mentions)
Sca 1 fitc, by BD (1 mentions)
Aria cell sorter, by BD (1 mentions)
Cd45 pe cytm7, by BD (1 mentions)
Rat igg isotype control pe, by BD (1 mentions)
Winmdi software, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Cd44 fitc, by Abcam (1 mentions)
17 protocols using cd133 pe
1
Quantifying Stem and Progenitor Cell Populations
2
Characterization of Murine Kidney Cell Populations
3
Immunophenotyping of Murine Cancer Cells
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Single cell suspension dissociated from S1M allografts and primary mouse cells were stained with Sca‐1 FITC (553335; BD Pharmingen), CD133 PE (12‐1331‐82; eBioScience), CD44 FITC (11‐0441‐81; eBioScience), CD45 PE‐CyTM7 (552848; BD Pharmingen), rat IgG isotype control PE (553930; BD Pharmingen), and rat IgG isotype control FITC (11‐4031‐81; eBioScience) for 1 h at 4°C in the dark room. The cells were analyzed on FACS Calibur (BD Biosciences) and sorted on an Aria cell sorter (BD Biosciences).
4
Characterization of Stem Cell Markers
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Cultured cells were detached in non-enzymatic cell dissociation medium (Sigma-Aldrich) to preserve cell membrane markers. Cells were incubated for 30 minutes at 4 °C with monoclonal antibodies against CD34-PE, VEGFR2-FITC, and CD133-PE (eBioscience) according to manufacturer’s protocol. Quantitative fluorescence analysis was performed using a fluorescence-activated cell sorting (FACS) flow cytometer and WinMDI software (Becton Dickinson).
5
Multiparameter Flow Cytometry Analysis
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Live cells were incubated with conjugated antibodies CD133-PE (1:100; 12-1338, eBioscience, San Diego, CA, USA) and CD44-FITC (1:50; ab19622, abcam, Cambridge, UK) at 4 °C for 30 min. PE Mouse IgG1 kapa (400111, Biolegend) and FITC Rat IgG2b (400633, Biolegend) were used as isotype controls. After washing with PBS, cells were re-suspended in 300 μl PBS and examined on an LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR, USA). For calcium detection, live cells were loaded with 5 μM Fluo3-AM (Sigma) for 45 min at 37 °C, washed with PBS, and checked on the flow cytometer. For cell cycle analysis, transfected cells were dissociated into single cell suspension and fixed in 70% ethanol. After washing with PBS, cells were treated with RNase (0.2 mg/ml) and stained in propidium iodide solution (10 μg/ml) and then analyzed by flow cytometry.
6
Mobilization of Endothelial Progenitor Cells
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To assess the endothelial progenitor cell (EPC) levels mobilized post-ischemic injury, peripheral blood samples were harvested at day 7 post-hindlimb ischemia in mice. Erythrocyte lysis buffer (BD Biosciences, Franklin Lakes, NJ, USA) was used to lyse red blood cells in a shaker at room temperature for 10 min. After fixing with 4% paraformaldehyde, the cells were stained with the antibodies CD34-FITC (11-0349-42, eBioscience, San Diego, CA, USA) and CD133-PE (12-1331-82, eBioscience). Stained cells were then analyzed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
7
Isolation and Identification of EpSCs
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Seven-to-nine-week-old male C57BL/6 (B6) mice were purchased from Hubei Provincial Center for Disease Control and Prevention (Wuhan, China). B6/SIRT2-/- mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China). EpSCs were isolated and identified as described previously [9 (link)]. In brief, after a laminectomy, spinal cord tissue was harvested and cut into 1 mm3 pieces, enzymatically dissociated in a solution containing 0.01% papain and 0.01% DNase I (Worthington, Biochemicals, NJ, USA) for 1~2 h at 37°C, and mechanically dissociated into a cell suspension. Then, the suspensions were incubated with an antibody mix containing CD133-PE (1:100, clone 13A4, eBioscience, CA, USA) and CD24-fluorescein isothiocyanate (FITC; 1:500, clone M1/69, BD Biosciences, CA, USA) for 30 min on ice. Cells were sorted with a FACSDiva flow cytometer (BD Biosciences).
8
Antibody Profiling for Cell Lineage
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Primary antibody against SOX9 (ab185230), Twist (ab49254), STAT3 (ab119352), p-STAT3 (ab30647 ), E-cad(ab181296), N-cad(ab76011), Vimentin (ab137321), ALDH1A1(ab52492), Ago2 (ab32381 ),GAPDH (ab8245) and actin(ab5694) were purchased from Abcam; MMP3 (Catalog,17873-1-AP) from Proteintech(Chicago, IL); CD133 (PE, eBioscience) from Invitrogen; IL-11 (Catalog Number. 220–11) from Peprotech. Secondary antibodies were purchased from Invitrogen. Pooled siRNAs against SOX9, mmu_circ_0000730, mmu_circ_0000363, mmu_circ_0000051, mmu_circ_0000551, and control siRNA were purchased from RiboBio (Guangzhou, China). Pooled biotin-labeled probes for RNA-pulldown against mmu_circ_0000730, mmu_circ_0000818, mmu_circ_0000551 and control oligos were purchased from RiboBio (Guangzhou, China). Inhibitors against mmu-let-7a-5p, mmu-let-7d-5p, mmu-let-7 g-5p, mmu-let-7i-5p, mmu-let-7 f-5p, mmu-let-7e-5p, mmu-miR-466i-3p and mmu-miR-466 f-3p, and control inhibitor were purchased from RiboBio (Guangzhou, China). PCR primers were purchased from RiboBio (Guangzhou, China). Probes for FISH against mmu_circ_0000730, mmu-miR-466i-3p were purchased from RiboBio (Guangzhou, China).
9
Multiparametric Flow Cytometric Analysis
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Flow cytometric analysis was performed on LD, HD, and LDHD using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences). 2 × 105 cells were suspended in staining buffer and incubated for 20 min at 4°C with fluorescently conjugated anti-human antibodies: CD90-FITC (Clone 5E10, BioLegend), CD105-PE (Clone SN6h, BioLegend), CD44-BV605 (Clone IM7, BioLegend), CD73-APC (Clone AD2, BioLegend), CD166-PerCP-eFluor™ 710 (Clone 3A6, Fisher Scientific), CD14-APC (Clone 61D3, eBioscience), CD45-VioBlue (Clone 5B1, Miltenyi), CD31-PE (Clone WM59, BD Biosciences), HLA-DR-PE-CF594 (Clone G46-6, BD Biosciences), CD10-APC (Clone HI10a, Biolegend), CD146-PE (Clone 541-10B2, Miltenyi Biotec), CD200-FITC (Clone OX104, Invitrogen), CD133-PE (Clone TMP4, eBioscience), and CD107-PerCP/Cy5.5 (Clone H4A3, BioLegend). Acquisition of 50,000 events for each cell sample was performed. Subsequent gating strategies were standardized for each sample based on scatter, singlets, and positive expression, which were overlaid with corresponding isotype controls.
10
Flow Cytometric Analysis of CSC Markers
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Sections of approximately 4-6 µm were cut and mounted on a pre-coated glass slide, following the procedure we reported previously 28 (link). Fixed cells were incubated in 1 mL 70% ethanol at -20 ℃ for at least 1 h. The cells were centrifuged at 500 ×g for 5 min, and suspended in 500-1000 µL PI staining buffer containing PBS, 50 µg/mL PI and 100 µg/mL RNAse A. For the CSC marker measurements, the cells were labeled with antibodies against CD133-PE, CD90-APC, EpCAM-PE-Cy7 (eBioscience), and the corresponding isotype antibody as a control to exclude nonspecific background staining. They were then incubated in the dark for 20-30 min and analyzed by FACSCalibur (Becton Dickinson, San Jos, CA).
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