LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit

Protocol tips

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	Add 1 μL of the reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes, protected from light.

Protocol tips
Add 1 μL of the reconstituted fluorescent reactive dye and incubate at room temperature or on ice for 30 minutes, protected from light.

Publication protocol

Preparation of embryonic mouse back and head skin for isolation of keratinocytes was performed as previously described (2). Briefly embryos and neonates: Skins were removed and treated with warm dispase for 1 hr at room temperature or overnight at 4ºC. The epidermis was peeled from the underlying dermis with forceps and placed into equal parts 0.25% Trypsin-EDTA and versene for 45 minutes at room temperature with shaking. E media was added to suspension to neutralize trypsin. Single-cell suspensions were obtained by filtering through a 70uM strainer and collected by centrifugation at 300g for 5 minutes. For detection of intracellular antigens including keratins, cells were washed in PBS and re-suspended at 1X10 cells/0.1mL and 0.9mL 100% ice cold MeOH was slowly added while vortexing to fix cells. Cells were placed at -20ºC for >30 minutes. Cell suspensions were incubated with the appropriate antibodies for 30 minutes on ice. The following antibodies were used for FACS: α6-integrin (eBiosciences), CD140a (eBiosciences), CD31 (eBiosciences), CD45 (eBiosciences), K5 (Fuchs Lab), K10 (Covance), DAPI or LIVE/DEAD Fixable Aqua was used to exclude dead cells. Native keratin expression was used to isolate differentiating populations for RNAi screen. Transgenic keratins driving fluorescent histone expression was used to isolate intact cells
for RNA extraction. Cell isolations were performed on FACS Aria sorters running FACS 6 Diva software (BD Biosciences). For EdU incorporation experiments, staining was performed using Click-iT EdU Alexa Fluor 647 Flow Cytometry Kit (Life Technologies) per manufacturer’s instructions. For cell cycle analysis MeOH fixed keratinocytes were stained with Dye Cycle Fx Violet (Invitrogen) per manufacturer instructions and cells were subsequently assayed. FACS analyses were performed using LSRII FACS Analyzers and results were analyzed with FlowJo vX software. Cells were prepared for ImageStream acquisition by fixation and staining just as they were prepared for FACS analysis.

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