As previously reported(8 (link)), ears or skin grafts were minced into small pieces and put into a gentle MACS C tube (Miltenyi) with 3 to 5 ml DMEM digestion solution containing 2% FCS, 10mM Hepes, 0.1mg/ml Liberase TM (Sigma), 0.1 mg/ml DNAse I (Sigma) and 0.5 mg/ml hyaluronidase (Sigma). After 1.5 hour shaking at 37°C, ear tissues were further broken down in a gentle MACS dissociator (Miltenyi) and debris were filtered. Cells were washed in FACS buffer and stained for flow cytometry. To separate the epidermis and dermis, ears were split into dorsal and ventral halves and incubated with 20 mM EDTA in PBS at 37°C for 2 hr. Then the separated epidermis and dermis were digested as above.
Protocol detail
Tissue Dissociation and Cell Isolation from Ear Samples
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Buffer
Cells
Dermis
Digestion 3
Dnase
Ears
Edta
Epidermis
Flow cytometry
Hepes
Hyaluronidase
Liberase
Skin grafts
Tissues
Corresponding Organization : Harvard University
Other organizations : University of Massachusetts Chan Medical School
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Variable analysis
independent variables
- Mincing ear or skin grafts into small pieces
- Digestion of ear or skin tissues in a gentle MACS C tube with DMEM digestion solution containing 2% FCS, 10mM Hepes, 0.1mg/ml Liberase TM, 0.1 mg/ml DNAse I, and 0.5 mg/ml hyaluronidase for 1.5 hours at 37°C
- Further breakdown of ear tissues in a gentle MACS dissociator
- Separation of epidermis and dermis by incubating ear halves with 20 mM EDTA in PBS at 37°C for 2 hours
- Cell characteristics and composition as measured by flow cytometry
- Use of FACS buffer for cell washing prior to flow cytometry
- Filtering of debris after tissue dissociation
- No positive or negative controls explicitly mentioned
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