Bulge cells and total keratinocytes were isolated from the dorsal skin of seven weeks-old Krt1–15-EGFP, Krt1–15-EGFP/K5CDK4, and Krt1–15-EGFP/CDK4−/− mice as previously described (Blanpain et al., 2004 (link)). Briefly, fat and underlying subcutis from the dorsal skins of mice were removed, and following trypsinization, neutralized cell suspensions were strained through 100uM and 40uM filters (BD Pharmigen, Franklin Lakes, NY). Single-cell suspensions in 2% FCS in PBS were incubated with primary antibodies for 30min. Primary antibodies used for FACS analysis were anti-α6 integrin (CD49f) (BD Pharmigen) directly coupled to PE-Cy5 (BD Pharmingen), and anti-CD34 (BD Pharmingen) coupled to biotin. Cells were further incubated with streptavidin coupled to specific fluorochromes for 30 min, washed, and resuspended in PBS with 2% FCS and propidium iodide (Sigma Aldrich, St. Louis, MO). Flow cytometry analysis (FACS) was conducted using a DAKO Cytomation MoFlo, in which cells were gated for single events/viability and sorted according to EGFP expression.
Anti α6 integrin cd49f
Manufactured by BD
Anti-α6 integrin (CD49f) is a monoclonal antibody that binds to the α6 subunit of the α6β1 and α6β4 integrins. Integrins are cell surface receptors that mediate cell-cell and cell-extracellular matrix interactions.
Automatically generated - may contain errors
Lab products found in correlation
Pe cy5, by BD (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Anti cd34, by BD (2 mentions)
Superscript 2 first strand synthesis kit, by Thermo Fisher Scientific (2 mentions)
Easysep mouse epithelial cell enrichment kit, by STEMCELL (2 mentions)
Epcam fitc conjugated antibody, by BioLegend (2 mentions)
Dneasy blood and tissue spin column kit, by Qiagen (2 mentions)
Rneasy spin column kit, by Qiagen (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Facsaria, by BD (2 mentions)
4 protocols using anti α6 integrin cd49f
1
Isolation and FACS Analysis of Keratinocyte Subpopulations
2
Isolation and Characterization of Bulge Cells
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Bulge cells and total keratinocytes were isolated from the dorsal skin of seven weeks‐old Krt1‐15‐EGFP, Krt1‐15‐EGFP/K5CDK4, and Krt1‐15‐EGFP/CDK4−/− mice as previously described (Blanpain et al., 2004 (link)). Briefly, fat and underlying subcutis from the dorsal skins of mice were removed, and following trypsinization, neutralized cell suspensions were strained through 100 μM and 40μM filters (BD Pharmigen). Single‐cell suspensions in 2% fetal calf serum (FCS) in phosphate‐buffered saline (PBS) were incubated with primary antibodies for 30 min. Primary antibodies used for FACS analysis were anti‐α6 integrin (CD49f) (BD Pharmigen) directly coupled to PE‐Cy5 (BD Pharmingen), and anti‐CD34 (BD Pharmingen) coupled to biotin. Cells were further incubated with streptavidin coupled to specific fluorochromes for 30 min, washed, and resuspended in PBS with 2% FCS and propidium iodide (Sigma Aldrich). Flow cytometry analysis (FACS) was conducted using a DAKO Cytomation MoFlo, in which cells were gated for single events/viability and sorted according to EGFP expression.
3
Enrichment and Sorting of Mammary Tumor Cells
4
Enrichment and Sorting of Mammary Tumor Cells
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Mammary tumors were dissociated into single cell suspensions through mechanical separation and enzymatic digestion as described22 (link). Dissociated tumor cells were enriched for Lin− (CD45−/ CD31−/ TER119−/ BP-1−) mammary epithelial cells with StemCell Technologies EasySep Mouse Epithelial Cell Enrichment Kits per the manufacturer’s instructions. Lin− cells were then incubated on ice for 20 min with anti-CD49f (α6 integrin) (BD Biosciences 555734) together with Alexafluor 647 (Invitrogen A21247) in PBS. Cells were spun down for 5 min at 550x g, then incubated with EpCAM-FITC conjugated antibody (Biolegend 118208) in PBS. Tumor cells were sorted on a BD FACS Aria cell sorter machine equipped with Diva software into their luminal (Lin−/ CD49flow/EpCAMhigh) and basal (Lin−/CD49fhigh/EpCAMlow) subpopulations. Sorted cells were collected into 15ml conical tubes containing PBS. Genomic DNA was collected from sorted cell populations using Qiagen Blood and Tissue DNeasy spin column kit. Total RNA was collected from sorted cell populations using Qiagen RNeasy spin column kit. RNA was reversed transcribed using Invitrogen Superscript II First Strand Synthesis kit.
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