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Cd49f

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CD49f is a cell surface antigen that is expressed on various cell types, including hematopoietic stem cells and certain epithelial cells. It functions as a receptor for the extracellular matrix protein laminin.

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Lab products found in correlation

Cd45ra, by BD (2 mentions) Propidium iodide, by Merck Group (2 mentions) Tryple, by Thermo Fisher Scientific (2 mentions) Pe cy7 strep, by BD (2 mentions) Lsr 2 facs machine, by BD (2 mentions) Aria 2, by BD (2 mentions) Cox 2, by Cayman Chemical (2 mentions) Dm7200, by Leica camera (2 mentions) Ptges2, by ABclonal (2 mentions) Ptges3, by ABclonal (2 mentions) Ptger2, by Abcam (2 mentions) F4 80, by BioLegend (2 mentions) Cd49f and cd24, by STEMCELL (1 mentions) Lsrii cytometer, by BD (1 mentions) Mitotracker orange cmtmros, by Thermo Fisher Scientific (1 mentions) Mitotracker green fm, by Thermo Fisher Scientific (1 mentions) Anti lineage cocktail, by BD (1 mentions) Facs caliber, by BD (1 mentions) Accutase, by BD (1 mentions) Cd326, by BD (1 mentions)

15 protocols using cd49f

1

Isolation of Mammary Stem and Cancer Stem Cells

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For isolation of MaSC and BCSCs, we used CD24lo and CD49fhi as markers for normal MaSC and CD44/CD24 double-positive for human BCSCs and CD49f/CD24 double-positive for mouse BCSCs as described in our previous manuscripts (15 (link), 21 (link)) and studies from other laboratories (22 (link), 23 (link)). We used the following antibodies for this study: CD49f and CD24 (STEMCELL Technology and eBioscience), CD45, CD31, CD49f, CD44 (BD bioscience), and Ter119 and CD61 (eBioscience). Blocking was done for 10 min with rat serum. Cells were stained for 30 min on ice and washed with staining media. Finally, cells were resuspended in staining media containing 7-aminoactinomycin D (1 μg/ml) or 4’−6-diamidino-2-phenylindole (DAPI, 1 μg/ml) to stain dead cells. Cells were sorted through Mo flow cell sorter and flow cytometry data was analyzed using LSR II and Flow-jo as described before in our manuscripts (15 (link), 21 (link)).
Bridges A.E., Ramachandran S., Pathania R., Parwal U., Lester A., Rajpurohit P., Morera D.S., Patel N., Singh N., Korkaya H., Manicassamy S., Prasad P.D., Lokeshwar V.B., Lokeshwar B.L., Ganapathy V, & Thangaraju M. (2020). RAD51AP1 deficiency reduces tumor growth by targeting stem cell self-renewal. Cancer research, 80(18), 3855-3866.
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2

Phenotypic Characterization of Murine and Human Hematopoietic Cells

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This was done using an LSRII cytometer or FACS Caliber (Becton Dickenson, San Diego, CA) and chromophore-conjugated antibodies used for mouse BM cell phenotyping (Mantel et al., 2010 (link); 2012 (link)). See also Figure S1C. Antibodies used for human phenotyping (Notta et al., 2011 (link)) were anti-lineage cocktail, CD34, CD38, CD45RA, CD90, and CD49f (BD Biosciences). For mitochondrial mass, membrane potential, and ROS analysis (Mantel et al., 2010 (link); 2012 (link)), we used Mitotracker Green FM, JC-1, and Mitotracker Orange CMTMRos respectively (Molecular Probes, Life Technologies; Grand Island, NY). CXCR4 antibodies were purchased from BD Biosciences. Phenotyping for mouse transplant chimerism/engraftment analysis was as noted (Mantel et al., 2012 (link); Broxmeyer et al., 2012 ). Flow cytometric analysis of apoptosis was assessed by activated caspase-3 (Mantel, et al., 2007 (link)).
Mantel C.R., O'Leary H.A., Chitteti B.R., Huang X., Cooper S., Hangoc G., Brustovetsky N., Srour E.F., Lee M.R., Messina-Graham S., Haas D.M., Falah N., Kapur R., Pelus L.M., Bardeesy N., Fitamant J., Ivan M., Kim K.S, & Broxmeyer H.E. (2015). Enhancing hematopoietic stem cell transplantation efficacy by mitigating oxygen shock. Cell, 161(7), 1553-1565.
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3

Immunophenotypic Characterization of MISB10 Cells

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MISB10 cells cultured in T75 flasks were treated with Accutase (BD Biosciences) and washed twice with PBS with 1% BSA. Suspensions were counted and measured for cell viability using the Vi-Cell XR cell counting and viability analyzer (Beckman Coulter). Cells were diluted in PBS to 1 × 106 cells/ml and stained for 30 min at RT with membrane viability dye (LIVE/DEAD Fixable Near-IR, Invitrogen). Cells were washed and distributed to a 96-well plate containing staining antibodies and Hoechst 33342 (Invitrogen). For characterization panels, immunophenotyping antibodies to the following targets were used: HER-2/neu, CD24, CD29, CD44, CD45, CD49f, CD90, CD166, CD326 (BD Biosciences), and CD133 (Miltenyi Biotec, Auburn, CA). Cells were incubated for 30 min at RT in the dark and then rinsed twice in PBS before acquisition with a BD LSRII flow cytometer. Analysis of results was performed using FACSDiva v6.1.3 and FlowJo analysis software (FlowJo).
Nykänen N., Mäkelä R., Arjonen A., Härmä V., Lewandowski L., Snowden E., Blaesius R., Jantunen I., Kuopio T., Kononen J, & Rantala J.K. (2021). Ex Vivo Drug Screening Informed Targeted Therapy for Metastatic Parotid Squamous Cell Carcinoma. Frontiers in Oncology, 11, 735820.
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4

Hematopoietic Stem Cell Immunophenotyping

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For model development, surface marker staining was performed with conjugated human antibodies: CD34, CD38, CD45RA, CD90, CD49f, CD10, CD135, CD7, CD3, CD19, CD41, CD235a, CD11b, CD16, CD33, CD14 (BD Biosciences). Surface marker staining for validation experiments was performed with conjugated human antibodies: CD34, CD45RA, CD90 (BD Biosciences). All samples were analyzed on a FACSCanto or FACS LSRFortessa flow cytometer (BD Biosciences).
Caldwell J., Wang W, & Zandstra P.W. (2015). Proportional-Integral-Derivative (PID) Control of Secreted Factors for Blood Stem Cell Culture. PLoS ONE, 10(9), e0137392.
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5

Characterization of Breast Cancer Stem Cells

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BrCSCs were exposed to primary antibodies CD44 (BU75, Ancell), CD24 (ML5, R&D System), CD10 (FR4D11, Santa Cruz Biotechnology), CD49f (GoH3 Miltenyi Biotec), EpCAM (AF960, R&D System) or corresponding isotype controls, rinsed and labeled with secondary antibodies. BrCSCs were stained with CD49f and CD24 and successively sorted via flow cytometry using an FACSAria cell sorter (BD Biosciences). The analysis of ALDH1 activity was performed using the ALDEFLUOR kit (StemCell Technologies). For cell cycle analysis, BrCSCs were fixed in 70% ethanol and incubated with 50 μg/mL propidium iodide (Sigma-Aldrich), 3.8 mmol/L sodium citrate (Sigma) and 10 μg/mL RNase (Sigma). Samples were analyzed by FACSCalibur and CellQuest Software (BD Biosciences).
Petrelli A., Carollo R., Cargnelutti M., Iovino F., Callari M., Cimino D., Todaro M., Mangiapane L.R., Giammona A., Cordova A., Montemurro F., Taverna D., Daidone M.G., Stassi G, & Giordano S. (2014). By promoting cell differentiation, miR-100 sensitizes basal-like breast cancer stem cells to hormonal therapy. Oncotarget, 6(4), 2315-2330.
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6

Characterizing Stem Cell Populations

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Before flow cytometry analysis or sorting, cells were dissociated as described above or washed and dissociated using TrypLE (Gibco, Carlsbad, CA, USA). Cells were resuspended in a 100μL staining volume of fluorescence activated cell sorting (FACS) buffer (Hanks' balanced salt solution + 0.1% bovine serum albumin) and kept on ice. The following antibodies were used: CD133/1, and CD133/2 biotin (Miltenyi Biotech, San Diego, CA, USA), CD90 (Biolegend, San Diego, CA, USA), CD15 (BD Bioscience, San Jose, CA, USA), CD49f (BD Bioscience, San Jose, CA, USA), AB25 (Miltenyi Biotech, San Diego, CA, USA) and PE-CY7 strep (BD Biosciences, San Jose, CA, USA). All analyses were conducted using a LSR II FACS machine (BD Biosciences, San Jose, CA, USA) or sorted on an ARIA-II (BD Biosciences, San Jose, CA, USA) at the Stanford University FACS facility. Appropriate isotype controls were used to control for nonspecific isotype background.
Nitta R.T., Gholamin S., Feroze A.H., Agarwal M., Cheshier S.H., Mitra S.S, & Li G. (2014). Casein Kinase 2α Regulates Glioblastoma Brain Tumor Initiating Cell Growth through the β-Catenin Pathway. Oncogene, 34(28), 3688-3699.
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7

Isolation and Characterization of Mesenchymal Stem Cells

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Bone marrow aspiration was approved by the Inha University Hospital Institutional Review Board (IRB) and written informed consent was obtained from a healthy donor (IRB number #10-51). Highly homogeneous clonal MSCs were isolated from BM as described previously (30 (link)). Markers of MSCs were determined by flow cytometry using a number of specific antibodies, including CD14 (BD Biosciences, San Diego, CA, USA), CD29 (Serotech, Kidlington, UK), CD31 (Serotec), CD34 (BD Biosciences), CD44 (Serotec), CD45 (BD Biosciences), CD49f (BD Biosciences), CD73 (BD Biosciences), CD90 (BD Biosciences), CD105 (Serotec), CD106 (BD Biosciences), CD133 (BD Biosciences), CD166(Serotec), HLA-DR (BD Biosciences), HLA-Class I (BD Biosciences), CXCR4 (BD Biosciences), and Oct-4 (Cell Signaling Technology, Danvers, MA, USA). MSCs were positive for CD29, CD44, CD49f, CD73, CD90, CD105, CD166, HLA-Class I, and Oct-4 but were negative for C14, CD31, CD34, CD45, CD106, CD133, CXCR4, and HLA-DR (data not shown). For the assessment of differentiation potential, adipogenic, osteogenic, and chondrogenic differentiations were induced as described (6 (link)). MSCs isolated were successfully differentiated into these 3 mesenchymal cell types, indicating the multilineage differentiation potential (data not shown).
Kim S.N., Lee H.J., Jeon M.S., Yi T, & Song S.U. (2015). Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells. Immune Network, 15(5), 241-251.
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8

Multiparametric Immunofluorescence Imaging

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Dorsal skin samples were embedded in OCT and sectioned at 8 µm thickness. Sections were fixed in formalin for 10 min, followed by two H2O washes (10 min each). Sections were blocked with blocking buffer (10% donkey serum in 1xPBST) for 1 h. Primary antibodies were diluted in blocking buffer and applied to the tissue and incubated overnight at 4 °C. Secondary antibodies were diluted in 1xPBST for 1 h at room temperature following three 1xPBST washes (5 min). Sections were washed in 1xPBST three times (5 min) and mounted with Fluoroshield with DAPI (Abcam, ab104139). Images were taken using the Leica DM7200 fluorescence imaging platform with LAS X version 3.7.5. Antibodies used: F4/80 1:600 (Biolegend, Cat#123101); Ly6G 1:600 (Biolegend, Cat#127601); CD3 1:800 (Biolegend, Cat#100201); Cox-2 1:600 (Cayman, Cat#160106); Ptges2 1:200 (Abclonal, Cat#A7137); Ptges3 1:200 (Abclonal, Cat#A5194); Ptger2 1:500 (Abcam, Cat#ab167171); Cd49f 1:100 (BD Biosciences, Cat#555734); Dct 1:600 (Abcam, Cat#ab221144), together with suitable Alex Fluor secondary antibodies (Abcam, Cat#ab150072, ab150149, ab150152, Fisher, Cat#A21207)
An L., Kim D., Donahue L.R., Mejooli M.A., Eom C.Y., Nishimura N, & White A.C. (2024). Sexual dimorphism in melanocyte stem cell behavior reveals combinational therapeutic strategies for cutaneous repigmentation. Nature Communications, 15, 796.
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9

Immunohistochemical Analysis of Skin Samples

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Immunochemistry and immunofluorescence were performed as described previously [44 (link)]. Skin samples were fixed in 4% PFA, paraffin-embedded 5-μm sections were stained with hematoxylin and eosin (H&E). For immunofluorescence staining, paraffin sections were microwave pretreated, and incubated with primary antibodies, then incubated with secondary antibodies (Invitrogen) and counterstained with DAPI in mounting media. The following antibodies and dilutions were used: K14 (rabbit, 1:1000, Covance), K14 (mouse, 1:500, Abcam),K5 (rabbit, 1:1000, Covance), Sox9 (rabbit, 1:100, Santa Cruz), AE13(mouse, 1:100, Abcam), AE15 (mouse, 1:100, Abcam), BrdU (rat, 1:200, Abcam), Cleaved Caspase-3 (rabbit, 1:100, Cell Signaling), Gata-3 (mouse, 1:100, Santa Cruz), K17 (rabbit, 1:100, Abcam), Foxn1 (mouse, 1:100, Santa Cruz), Dlx3 (rabbit, 1:100, Santa Cruz), Sostdc1 (rabbit, 1:100, Abcam), Hoxc13 (mouse, 1:100, Sigma), Lef1 (rabbit, 1:100, Cell Signaling), Ki67 (mouse, 1:100,Novacastra), Bmpr1a (rabbit, 1:100, Abcam), Msx2 (rabbit, 1:200, Santa Cruz), k15 (mouse, 1:150, Vector Labs), CD49f (rat,1:100,BD), p63 (mouse, 1:200, Santa Cruz), K10 (rabbit, 1:1000, Covance), Loricrin (rabbit, 1:500, Covance), K1 (rabbit, 1:500, Covance).
Yuan S., Li F., Meng Q., Zhao Y., Chen L., Zhang H., Xue L., Zhang X., Lengner C, & Yu Z. (2015). Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22. PLoS Genetics, 11(5), e1005253.
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10

Characterizing Stem Cell Populations

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Before flow cytometry analysis or sorting, cells were dissociated as described above or washed and dissociated using TrypLE (Gibco, Carlsbad, CA, USA). Cells were resuspended in a 100μL staining volume of fluorescence activated cell sorting (FACS) buffer (Hanks' balanced salt solution + 0.1% bovine serum albumin) and kept on ice. The following antibodies were used: CD133/1, and CD133/2 biotin (Miltenyi Biotech, San Diego, CA, USA), CD90 (Biolegend, San Diego, CA, USA), CD15 (BD Bioscience, San Jose, CA, USA), CD49f (BD Bioscience, San Jose, CA, USA), AB25 (Miltenyi Biotech, San Diego, CA, USA) and PE-CY7 strep (BD Biosciences, San Jose, CA, USA). All analyses were conducted using a LSR II FACS machine (BD Biosciences, San Jose, CA, USA) or sorted on an ARIA-II (BD Biosciences, San Jose, CA, USA) at the Stanford University FACS facility. Appropriate isotype controls were used to control for nonspecific isotype background.
Nitta R.T., Gholamin S., Feroze A.H., Agarwal M., Cheshier S.H., Mitra S.S, & Li G. (2014). Casein Kinase 2α Regulates Glioblastoma Brain Tumor Initiating Cell Growth through the β-Catenin Pathway. Oncogene, 34(28), 3688-3699.
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