I would like to use flow cytometry to characterize the cells of a Single Cell solution of human skin samples.
Does anyone have a tip which marker is suitable for keratinocytes?
Thanks!!
Hello,
You could use Keratins 5 and 15 for basal keratinocytes, and Keratins 2 and 10 for suprabasal keratinocytes.
Please refer to the article below for more information.
https://doi.org/10.1369/jhc.2008.951194
Best.
Dear Manuel!
Please You look at the article:
Article α6 Integrin and CD44 Enrich for a Primary Keratinocyte Popul...
Cell Culture
Primary human keratinocytes were isolated from redundant facelift skin and passage 2–3 cells were used in all experiments unless otherwise stated. Keratinocytes were grown on mitomycin-treated 3T3 feeder layers in growth factor-containing medium (DMEM-F12 modified medium, PAA, Yeovil, UK) supplemented with 10% foetal bovine serum (FBS) (Biowest, East Sussex, UK), 1% (vol/vol) glutamine, 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, 5 µg/ml transferrin, 10−10 M cholera toxin, 0.013 µg/µl lyothyronine (Sigma, Poole, UK) and 10 ng/ml EGF (Serotec, Oxford, UK) at 37°C in 10% CO2.
Flow Cytometry
Cells were grown at clonal density until they reached 60% confluence (5 days), washed with PBS and removed from the culture dish using trypsin-EDTA (PAA, Yeovil, UK). Following neutralisation and washing with PBS the cells were resuspended at 1×106/ml in PBS/1% FBS with 5×105 cells used per incubation and analysis. Incubation was at 4°C for 30 min using CD44-FITC and CD49f-PE-Cy5 conjugated antibodies (BD Bioscience, Oxford, UK). Simultaneously cells were incubated with the corresponding IgG isotype controls (BD Bioscience, Oxford, UK). Cells were then washed in PBS/1% FBS and either recorded as live cells on a BD LSRII FACS machine or fixed cells following treatment with 1% paraformaldehyde. FACSDiva software was used to analyse all samples, with isotype and single-colour negative controls used to establish compensation settings, and a minimum of 30,000 events recorded per sample. For sorting of the cells a BD FACSAria machine was used. Other live cell-surface flow cytometry analysis used the BD antibodies β1 integrin (CD29-APC), transferrin receptor (CD71) with biotynilated-PE secondary, and appropriate IgG isotype controls (BD Bioscience, Oxford, UK). The FITC, PE and PE-Cy5 antibodies were excited using an argon laser (488 nm) and collected using a 530/30 nm filter (FITC), 575/26 nm filter (PE), and 675/20 nm filter (PE-Cy5). The APC antibody was excited using the 633 nm red diode and collected using a 660/20 nm filter.
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