The cell spheres were induced and then isolated from the Ishikawa cells as described above. The single-cell suspension (100 µl of buffer per 107 cells) was prepared and stained with PE-CD133 and PE-Cy7-CD44 (BD, Franklin Lakes, NJ, USA) for 30 min in the dark at 4 °C for FACS sorting on AriaIII (BD). The obtained CD44+/CD133+ cells were cultured in stem cell media as described previously.
Cd44 pe cy7
Manufactured by BD
Sourced in United States
CD44-PE-Cy7 is a fluorescently-labeled monoclonal antibody that binds to the CD44 cell surface glycoprotein. CD44 is a receptor for hyaluronic acid and is involved in cell-cell interactions, cell adhesion, and migration. The PE-Cy7 fluorescent label allows for detection and analysis of CD44-expressing cells using flow cytometry or other fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cd62l pe, by BD (3 mentions)
Cd133 pe, by BD (2 mentions)
Facsaria, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Cd25 apc, by BD (2 mentions)
Cd62l apc, by BD (2 mentions)
Ifn γ pe, by BD (2 mentions)
Foxp3 fitc, by BD (2 mentions)
T bet pe, by BD (2 mentions)
Cd278 icos biotin, by BD (2 mentions)
Rorγt pe, by BD (2 mentions)
Cd25 bb515, by BD (2 mentions)
Live dead fixable near ir stain, by Thermo Fisher Scientific (2 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (2 mentions)
Canto 2, by BD (2 mentions)
Cd25 pe, by BD (2 mentions)
Cd3 fitc, by BD (2 mentions)
Aria 3, by BD (1 mentions)
Isotype control antibody, by BD (1 mentions)
Trypsin edta, by Solarbio (1 mentions)
23 protocols using cd44 pe cy7
1
Isolation and Culture of CD44+/CD133+ Cells
2
Isolation of Putative Endometrial Cancer Stem Cells
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Ishikawa cells were suspended cultured in stem cell culture medium for 24 h, the suspended cells were digested into single cells with trypsin-EDTA (0.25%, Solarbio, Beijing, China). The single cell suspension (100 µL buffer/107 cells) was incubated with PE-CD133, PE-Cy7-CD44, and isotype control antibodies (BD, Franklin Lakes, NJ, USA) at 4 °C in the dark for 30 min, and the cells were sorted by flow cytometry (FACSARia, BD, USA). The CD44+/CD133+ cells obtained through sorting were considered as ECSCs, and other cells were non-stem cells.
3
Comprehensive Immune Cell Profiling
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Flow cytometry analyses were performed on a LSR Fortessa (BD), and cell sorting was performed on a FACSAria II (BD). For HLA detection on immune cells, the following antibodies were used: Mouse anti-human V450 CD4 (BD, 560345), APC-Cy7 CD8a (Tonbo, 25–0088-T100), BV421 CD56 (BioLegend, 318328), FITC CD19 (eBioscience, 11–0199-42), BV510 HLA-ABC (BioLegend, 311436), PE-Cy7 HLA-DR (eBioscience, 25–9952-42), and Alx647 HLA-DQ (BD, 564806). To detect HLA-DP, cells were labeled with mouse anti-human HLA-DP (Abcam, AB20897), washed and stained with PE goat anti-mouse IgG (eBioscience, 12–4010-82). For T cell phenotyping, the following antibodies were used: Mouse anti-human PE CD69 (BD, 555531), PE-Cy7 CD44 (BD, 560533), BV421 CD25 (BD, 562443), PE-Cy7 PD-1 (BioLegend, 329918), and BV421 TIGIT (BD, 747844).
4
Immunophenotypic Analysis of Leukemic Engraftment
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CD45-APC (BD, San Jose, CA, USA; 555485), CD33-PE (BD 555450), CD44-FITC (BD 555478), CD44-PE-Cy7 (BD 560533), CD45.1-PerCP-Cy5.5 (BD 560580), CD34-APC-Cy7 (Biolegend, Ozyme, Saint Quentin Yvelines, France; 343514), Lin1-FITC (BD 340546), CD123-PE (BD 340545), CD45RA-Alexa Fluor 700 (BD 560673) and CD38-APC (BD 555362) fluorescent antibodies were used to analyze leukemic cells before and after injection into animals to determine phenotypic analysis of engrafted cells and percentage of leukemic cell engraftment. Absolute cell counts were determined with CountBright absolute counting beads (Invitrogen) following the manufacturer's recommendations.
5
Quantifying CD44 Expression by Flow Cytometry
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CD44 surface marker expression was determined by flow cytometry. Samples were prepared as previously described [5 (link)] and analyzed on a FACS Canto II. CD44 PE-Cy7 and PE-Cy7 Mouse IgG2b, κ (BD Biosciences) were used as primary antibody and isotype control, respectively. Cell viability was assessed by staining dead cells with 1 μM Sytox blue (Life technologies).
6
Multiparametric Flow Cytometry Analysis
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Cell surface antibody and tetramer staining was performed concurrently in FACS buffer for 1 hr on ice in the dark, washed and fixed. To stain for intracellular cytokines, samples from in vitro restimulated cultures were permeabilize and stained with cytokine antibodies. Samples were washed and resuspended and FACS analyzed. Flow cytometry data were acquired on BD LSRII and analyzed with FlowJo software (Tree Star, Ashland, OR).
Mouse specific antibodies were purchased from eBioscience (San Diego, CA): CD44-PE Cy7, IFN-γ-eFluor450, CD8α-Alexa Fluor 700, TCRβ-PerCP-Cy5.5, KLRG1-APC, Thy1.1-eFluor 450 or from BD Bioscience: CD62L-APC-eFluor780 and CXCR3-FITC. PE-streptavidin-biotin SVLAFRRL: H-2Kb tetramers were prepared as previously described (Wilson et al., 2010 (link)).
Mouse specific antibodies were purchased from eBioscience (San Diego, CA): CD44-PE Cy7, IFN-γ-eFluor450, CD8α-Alexa Fluor 700, TCRβ-PerCP-Cy5.5, KLRG1-APC, Thy1.1-eFluor 450 or from BD Bioscience: CD62L-APC-eFluor780 and CXCR3-FITC. PE-streptavidin-biotin SVLAFRRL: H-2Kb tetramers were prepared as previously described (Wilson et al., 2010 (link)).
7
Comprehensive Bone Marrow Flow Cytometry
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Flow cytometry was performed on total bone marrow or thymic single cell suspensions. Cells were stained with fluorophore-conjugated antibodies from eBioscience: CD4-Alexa Fluor 700 (56-0041), CD4-PE (12-0041), CD8-eFluor 450 (48-0081), CD24-PE (12-0241), CD25-APC (17-0251), CD34-Alexa Fluor 700 (56-0341), CD44-PE-Cy7 (25-0441), c-Kit-APC-eFluor 710 (47-1171), Flt3-PE (12-1351), IL-7Rα-eFluor 450 (48-1271), Sca1-APC (17-5981) or BD Pharmingen: Lineage Cocktail-APC (558074), Lineage Cocktail-PerCP-Cy5.5 (561317), and TCRβ-APC (561080). Samples were analyzed on an LSR Fortessa flow cytometer (BD Biosciences) in the Flow Cytometry and Cell Sorting Core at BCM. Data interpretation used FACSDiva (BD Biosciences) and FlowJo (TreeStar Inc.) software.
8
Immunophenotyping of Cell Adhesion Molecules
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Cells were harvested with 0.01 M EDTA/PBS, blocked with human IgG (0.1 mg/ml in RPMI media (Life Technologies), 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA), 0.1% sodium azide (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min on ice prior to staining with an antibody master mix (CD44-PECy7, CD62P-APC, CD62E-APC, ICAM1-PE, ICAM2-APC, VCAM1-FITC, JAMA-PE (all BD), JAMC-VioBright FITC (Miltenyi Biotec, Bergisch Gladbach, Germany)) or the respective isotype controls (IgG2-PECy7, IgG1-APC, IgG1-PE, IgG1-FITC (all BD Biosciences, Franklin Lakes, NJ, USA), REA Control VioBright FITC (Miltenyi Biotec)) with viability dye 7-AAD (BD) in RF10 at manufacturer’s recommended concentrations for 30 min, 4°C in the dark. Cells were washed, fixed with FACS fix (PBS with 1% paraformaldehyde, 20 g/L glucose and 5 mM sodium azide (all Sigma-Aldrich, St Louis, MO, USA)) and analyzed on the C6 Accuri™ flow cytometer (BD) using the CFlow® Plus software (BD Biosciences) and FCS Express 4 Flow Cytometry: Research Edition software (De Novo, Pasadena, CA, USA).
In similar experiments, ECFCs and C32 melanoma cells were treated without or with 50 ng/ml VEGF-A (Sigma) for 72 h with the final 24 h including (or not) 10 ng/ml TNFα (R&D Systems). Cells were then assessed for cell surface expression of ICAM-1 and cell viability as detailed above.
In similar experiments, ECFCs and C32 melanoma cells were treated without or with 50 ng/ml VEGF-A (Sigma) for 72 h with the final 24 h including (or not) 10 ng/ml TNFα (R&D Systems). Cells were then assessed for cell surface expression of ICAM-1 and cell viability as detailed above.
9
Multiparametric Flow Cytometry Analysis
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Surface staining was performed with directly conjugated monoclonal antibodies for 20 min at room temperature for human samples. The cells were washed and resuspended in PBS before flow cytometric analysis. The monoclonal antibodies used were anti‐human CD4–Percp‐Cy5.5/APC‐H7, CD8–APC (allophycocyanin)‐R700/V500, PD‐1–PE (phycoerythrin)–Cy7, TIM‐3–APC, TIGIT–BV605, 2B4–AF700, and CD160–PE (BD Bioscience San Diego, CA, USA). Intracellular staining was carried out by using a fixation/permeabilization kit (BD Bioscience) after resuspension according to the manufacturer's instructions. Ki67–PE (BD Pharmingen) was added and incubated for 20 min at room temperature.
The surfaces of mouse samples were stained with direct‐conjugated monoclonal antibodies for 30 min at 4 °C. After incubation, the cells were washed and resuspended in phosphate‐buffered saline before flow cytometry analysis. The monoclonal antibodies used were anti‐mouse CD3–Percp, CD4–APC‐H7, CD8–FITC (fluorescein), CD44–PE–Cy7, and CD62L–APC (BD Bioscience San Diego, CA, USA). Detailed antibody information was listed in TableS3 (Supporting Information).
The surfaces of mouse samples were stained with direct‐conjugated monoclonal antibodies for 30 min at 4 °C. After incubation, the cells were washed and resuspended in phosphate‐buffered saline before flow cytometry analysis. The monoclonal antibodies used were anti‐mouse CD3–Percp, CD4–APC‐H7, CD8–FITC (fluorescein), CD44–PE–Cy7, and CD62L–APC (BD Bioscience San Diego, CA, USA). Detailed antibody information was listed in Table
10
Flow Cytometry Analysis of Cell Surface Markers
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All of the anti-human antibodies used in this study, including HER2 (phycoerythrin, PE), CD3 (chlorophyll protein complex PerCP), CD4 (fluorescein isothiocyanate, FITC), CD8-PE, CD45RO (allophycocyanin, APC), CD56-APC, CD62L-PE, CCR7-PE-Cy7, and CD44-PE-Cy7, were purchased from BD Biosciences. Isotype-matched control mAbs were applied in all the procedures. Additionally, GFP-positive HER2KD N87 and 7901 cells were separated from untransfected cells, based on the degree of fluorescence, by a BD Influx cell sorter (BD Biosciences). FACS data were analyzed by a FACSCalibur flow cytometer (BD Biosciences) and FlowJo software (Version 10.0.7, FlowJo, Ashland, OR).
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