Tail skin was removed from the tail bone and incubated overnight at 4°C in HBSS (Gibco) and 0.25% trypsin (Gibco).The epidermis was then separated from the dermis and incubated on a rocking plate (100 rpm) at room temperature for 5 min. Basal cells were mechanically separated from the epidermis by flushing 10 times under the epidermis. Tissues were then cut into small pieces with a scalpel and incubated again for 5 min on a rocking plate (100 rpm) at room temperature. Trypsin was then neutralized by adding DMEM (Gibco) supplemented with 5% Chelex-treated FCS, and the cells were mechanically separated by pipetting 10 times and filtrated on a 70-μm filter (Falcon). Cells were incubated in 2% FCS/PBS with primary antibodies for 30 min on ice. Cells were washed with 10 ml of 2% FCS/PBS and then incubated for 30 min in APC (allophycocyanin)–conjugated streptavidin (BD Biosciences), and then washed again and resuspended in 200 μl of 2% FCS/PBS with Hoechst (10 mg/ml) diluted at 1:4000. Living epidermal cells were gated by forward scatter, side scatter, and negative for Hoechst. Basal interfollicular epidermis keratinocytes were stained using fluorescein isothiocyanate–conjugated anti–α6 integrin (clone GoH3, 1:200, eBioscience, catalog number MAB1378) and biotinylated CD34 (clone RAM34, 1:50, BD Biosciences, catalog number 13-0341), followed by avidin/APC-streptavidin used to stain and exclude hair follicle stem cells. Basal keratinocytes were isolated on the basis of α6 integrin expression with exclusion of CD34-positive cells. In Vegfa overexpression conditions, basal keratinocytes expressing the transgene are GFP positive, and we labeled basal keratinocytes using PE-conjugated anti–α6 integrin (clone GoH3, 1:200, eBioscience, catalog number MAB1378), and bulge cells were labeled with biotinylated CD34 (clone RAM34, 1:50, BD Biosciences). We used PE-conjugated anti-CD45 (clone 30-f11, 1:200, eBioscience, catalog number 12-0451/553081), PE-conjugated anti-CD31 (clone MEC13.3; 1:100, BD PharMingen, catalog number 550274), and PE-conjugated anti-Pdgfrα (clone APA-5, 1:200 eBioscience, catalog number12-1401/624049) to exclude dermal cells and avoid dermal contamination, and anti-mouse/human CD11b (clone M1/70, BioLegend), anti-mouse CD16/32 (clone 93, BioLegend), anti-mouse Ly6C (HK1.4, BioLegend), and anti-mouse Ly6G (1A8, BioLegend) were used to gate for neutrophils, macrophages, and monocytes. FACS analysis was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences). Sorted cells were collected into lysis buffer for RNA extraction.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.