Dissociated cells were resuspended at 1×106 cells/ml in DMEM + 2% FBS prior to the addition of MIC1-1C3 hybridoma supernatant (at a 1:20 dilution) or a 1:200 dilution of purified MIC1-1C3 antibody (Novus) and incubation at 4°C (for 30’). After a wash with cold DPBS, cells were resuspended in DMEM + 2% FBS containing a 1:200 dilution of APC-conjugated goat anti-rat secondary antibody adsorbed against mouse serum proteins (Jackson Immunoresearch). After another wash, cells were resuspended in DMEM + 5% rat serum (Serotec) and held on ice (10’) to block the secondary antibody. A final incubation with FITC-conjugated anti-CD26 (BD Biosciences, Franklin Lakes, NJ), PE-conjugated anti-CD133 (eBioscience, San Diego, CA) and PE-Cy7-conjugated anti-CD45 (BD Biosciences) + anti-CD11b/Mac1 (BD Biosciences) + anti-CD31 (Abcam, Cambridge, MA) facilitated cell subfractionation and exclusion gating of hematopoietic (CD45+, CD11b+) and endothelial (CD31+) cells. Propidium iodide staining was used to label dead cells for exclusion. Cells were analyzed and sorted with a Cytopeia inFluxV-GS (Becton-Dickenson, Franklin Lakes, NJ); FSC : Pulse-width gating was used to exclude cell doublets from analysis and collection.
Pe conjugated anti cd133
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PE-conjugated anti-CD133 is a monoclonal antibody that binds to the CD133 antigen. CD133 is a marker of hematopoietic stem cells and some types of cancer stem cells. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD133-positive cells using flow cytometry or other fluorescence-based methods.
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Lab products found in correlation
Pe cy7 conjugated anti cd45, by BD (1 mentions)
Cytopeia influxv gs, by BD (1 mentions)
Anti cd11b mac1, by BD (1 mentions)
Anti cd31, by Abcam (1 mentions)
Apc conjugated anti cd11b, by BioLegend (1 mentions)
Fitc conjugated anti cd31, by BioLegend (1 mentions)
Ebm 2, by Lonza (1 mentions)
Anti ifn γ, by BD (1 mentions)
Anti granzyme b, by BD (1 mentions)
Percp conjugated anti cd3, by BD (1 mentions)
Pe conjugated anti cd8, by BD (1 mentions)
Anti perforin, by BD (1 mentions)
Fitc conjugated anti cd44, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Fitc conjugated anti cd34, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Fitc conjugated anti cd44, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Trypsin, by Corning (1 mentions)
5 protocols using pe conjugated anti cd133
1
Multiparameter Flow Cytometry for Cell Subpopulation Identification
2
Isolation and Culture of Endothelial Progenitor Cells
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Bone marrow cells were collected and cultured as aforementioned. Cell aliquots (1×106) were incubated for 20 min at 4°C with the following anti-mouse antibodies: APC-conjugated anti-CD11b (dilution, 1:100; BioLegend, Inc.), FITC-conjugated anti-CD31 (cat. no. 102506; dilution, 1:50; BioLegend, Inc.), Per CP-conjugated anti-CD144 (cat. no. 46-1441-82; dilution, 1:50; eBioscience; Thermo Fisher Scientific, Inc.) and PE-conjugated anti-CD133 (cat. no. 141203; dilution, 1:40; BioLegend, Inc.). Acquisition was performed using a FACSAria flow cytometer, and data were analyzed using FACSDiva software version 6.1.3. Sorted CD133+/CD31+/CD144+/CD11b− cells were enriched by further culture in endothelial growth basal medium (EBM)-2 (Lonza Group, Ltd., Basel, Switzerland).
3
Multicolor Flow Cytometry Analysis
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FITC-conjugated anti-CD44, PerCP-conjugated anti-CD3, PE-conjugated anti-CD8, FITC-conjugated anti-Granzyme A, anti-Granzyme B, anti-perforin and anti-IFN-γ were all purchased from BD Pharmingen. PE-conjugated anti-CD133 was purchased from eBiosciences. For cell surface staining, SMMC7721 and HepG2 cells were resuspended in 100μl staining buffer containing 10% FBS and put on ice for 20 min to block Fc receptors, then incubated with FITC-conjugated anti-CD44and PE-conjugated anti-CD133or isotype control for 30 min. The cells were then washed with 1ml ice-cold staining buffer for 2 times and centrifuged (300g) at 4°C for 5 min. The collected cells were suspended in 500μl staining buffer solution. PBMCs were incubated with SMMC7721 cells for 5 hours with brefeldin A at a final concentration of 10μg/ml, then collected and washed. As discussed above, PerCP-conjugated anti-CD3 and PE-conjugated anti-CD8 were used for surface staining. After that, cells were fixed and permeabilized, and then intracellular staining of FITC-conjugated anti-GranzymeA, Granzyme B, perforin and IFN-γ was performed. All samples after staining were evaluated using BD FACS Canto II with Diva software and analyzed using Flowjo 7.6.5.
4
Lung Tissue Cell Characterization
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Suspended BM non-red blood cells and single-cell suspensions freshly obtained from collagenasedigested lung tissues were labeled with FITCconjugated anti-CD34 (560238, BD Biosciences, USA), PE-conjugated anti-CD133 (12-1331, eBioscience, USA), and APC-conjugated anti-Flk-1 (560070, BD Biosciences, USA) at 4oC for 30 min in the dark. Approximately, 5×105 (link) cells were analyzed by flow cytometry for each experiment using a FACSCaliburTM flow cytometer (BD Biosciences, USA).
5
Isolation and Characterization of Tumor Cells
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Adherent cells were detached with 0.05% Trypsin (Corning, Manassas, VA), washed and re-suspended in phosphate buffered saline (PBS). Tumors were mechanically and enzymatically disaggregated into single-cell suspensions, as in [35 (link)]. 1×106 cells were incubated with PE-conjugated anti-CD133 and/or FITC-conjugated anti-CD44 (eBioscience, San Diego, CA; 12-1338-42 and 11-0441-82). Labeled cells were analyzed by LSR Fortessa Cell Analyzer (BD Bioscience). Data was analyzed with Flowjo software. At least three samples were analyzed in triplicate and a normal distribution was observed for biological replicates.
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