Dermis was fragmented using scissors and digested using a stirring solution of 2 mg/mL of Type I collagenase (GibcoTM) in Dulbecco–Vogt modified Eagle medium (GibcoTM) for 20 h at 37 °C. The digested tissue was filtered using a Cell Strainer of 100 µm (Fisherbrand®), neutralized using fibroblast medium and centrifuged (20 min/24 °C/450× g) [11 (link)]. Finally, dermal cells were counted and seeded at 115,000 cells/cm2 in fibroblast medium. This primary culture was named passage 0 (P0). The estimated time and cost (considering only the price of the enzymes) for the dermal cell isolation were 22 h and USD 11.00, respectively.
Cell strainer of 100 m
Manufactured by Thermo Fisher Scientific
The Cell Strainer of 100 μm is a laboratory equipment used for filtration and separation of cells. It features a 100-micron mesh size for efficient cell isolation and purification.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (2 mentions)
Gentamicin, by Gemini Bio (2 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Dulbecco vogt modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Isoproterenol hydrochloride, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hyclone fetalclone 2 serum, by GE Healthcare (1 mentions)
Ham s f12, by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Adenine, by Merck Group (1 mentions)
Tryple select enzyme 10x, by Thermo Fisher Scientific (1 mentions)
4 protocols using cell strainer of 100 m
1
Isolation of Dermal Fibroblasts from Skin
2
Isolation of Epithelial Cells from Tissue
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Fragments of the epithelium were incubated for 25 min at 37 °C in 20 mL of a 0.05% Trypsin (GibcoTM, Waltham, MA, USA)/0.01% EDTA stirring solution (J.T. Baker, Phillipsburg, NJ, USA). Then, cell suspension was neutralized, filtered using a Cell Strainer of 100 µm (Fisherbrand®) and centrifuged (10 min/24 °C/300× g). Finally, epithelial cells were counted, seeded at 40,000 cells/cm2 on a feeder layer of irradiated human fibroblasts (8000 cells/cm2) and cultured in keratinocyte medium (Dulbecco–Vogt modified Eagle medium (GibcoTM): Ham’s F12 (GibcoTM), ratio 3:1, supplemented with 24.25 μg/mL adenine (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 0.4 μg/mL hydrocortisone (Galenova, Saint-Hyacinthe, QC, Canada), 0.212 μg/mL isoproterenol hydrochloride (Sigma-Aldrich), 5% bovine HyClone FetalClone II serum (GE Healthcare, Chicago, IL, USA), 10 ng/mL human epidermal growth factor (Austral Biologicals, San Ramon, CA, USA), 100 U/mL penicillin (Sigma-Aldrich) and 25 μg/mL gentamicin (Gemini Bio)) [9 (link),10 (link)]. This primary culture was named passage 0 (P0). Including the thermolysin digestion step, the estimated time and cost (considering only the price of the enzymes) for the epithelial cell isolation were 18 h and USD 21.00, respectively.
3
Isolation of Epidermal Keratinocytes
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Epidermis was mechanically fragmented into small pieces using scissors and incubated for 15 min in a stirring 5 mL of TrypLE Select Enzyme 10X (GibcoTM) at 37 °C (8 cycles). After each cycle, digested solution containing the cells and pieces of tissue was filtered using a Cell Strainer of 100 µm (Fisherbrand®). The filtered cell suspension was neutralized using 10 mL of keratinocyte medium and kept while remaining tissue was digested again. After the 8th cycle, the cell suspension was centrifuged (10 min/24 °C/300× g) [11 (link)]. Cells were counted and seeded at 130,000 cells/cm2 on a feeder layer of irradiated human fibroblasts (8000 cells/cm2) and cultured in keratinocyte medium. This primary culture was named passage 0 (P0). The estimated time and cost (considering only the price of the enzymes) for the epithelial cell isolation were 5 h and USD 81.00, respectively.
4
Isolation of Dermal Cells from Tissue
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Dermal tissue was digested for 3 h at 37 °C by stirring in 20 mL of a solution constituted of 0.125 U/mL Type H collagenase (Roche, Basilea, Switzerland) diluted in fibroblast medium (Dulbecco–Vogt modified Eagle medium (GibcoTM) supplemented with 10% Avantor Seradigm FB Essence (Avantor®, Radnor, PA, USA), 100 U/mL penicillin (Sigma-Aldrich) and 25 μg/mL gentamicin (Gemini Bio)). After incubation, cell suspension was filtered using a Cell Strainer of 100 µm (Fisherbrand®), separated into four 50 mL tubes (5 mL/tube) and digestion was neutralized with 45 mL/tube of fibroblast medium. After centrifugation of the cell suspensions (10 min/24 °C/450× g), the supernatant was discarded until 10 mL were left in each tube; pellets were resuspended and combined to have two tubes with 20 mL in each. These were centrifuged (10 min/24 °C/450× g) and previous steps were repeated once to obtain one tube with 20 mL of cell suspension. Finally, this tube was centrifuged (10 min/24 °C/450× g). Dermal cells were counted and seeded at 40,000 cells/cm2 in fibroblast medium [9 (link),10 (link)]. This primary culture was named passage 0 (P0). Including the thermolysin digestion step, the estimated time and cost (considering only the price of the enzymes) for the dermal cell isolation were 21 h and USD 19.50, respectively.
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