WT primary mouse keratinocytes were isolated from neonatal mouse skin as previously described 11 (link). To test the biologic activity and ability of Tat-Rac1 to enter into cells, mouse keratinocytes, human HaCaT keratinocytes (from Thermo Fisher Scientific), or human dermal fibroblasts (ATCC, Manassas, VA) were cultured on the 8-well chamber slides for 12h, then Tat-Rac1 protein (1μg/ml) or BrdU (10μM) was added to the culture medium and incubated for 3h; slides were fixed in cold methanol for 5min and washed in PBS. V5 or BrdU were stained using anti-V5 or anti-BrdU antibodies. Cell proliferation response to Tat-Rac1 protein was evaluated by the percent of BrdU labelled cells in total counted cells. Each experiment was averaged from 8-well chambers; data from 3 separate experiments as mean ± SD.
Hacat human keratinocytes
Manufactured by Thermo Fisher Scientific
Sourced in United States
HaCaT human keratinocytes are a spontaneously immortalized human keratinocyte cell line. They are derived from normal human skin and maintain characteristics of undifferentiated keratinocytes.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Human dermal fibroblasts, by American Type Culture Collection (1 mentions)
Tcf lef luciferase reporter plasmid, by Promega (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Epilife, by Thermo Fisher Scientific (1 mentions)
293t cell, by American Type Culture Collection (1 mentions)
Ly294002, by Merck Group (1 mentions)
Follicle dermal papilla cell growth medium kit, by PromoCell (1 mentions)
Human melanocyte growth supplement, by Thermo Fisher Scientific (1 mentions)
Medium 254, by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Welgene (1 mentions)
B16f0 mouse melanoma cells, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Welgene (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Caski, by American Type Culture Collection (1 mentions)
5 protocols using hacat human keratinocytes
1
Evaluating Tat-Rac1 Protein in Keratinocytes
2
Human Hair Follicle Dermal Papilla Cell Culture
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Human hair follicle dermal papilla (HDP) cells were purchased from PromoCell (Germany). The passage 3-7 cells were maintained in 5% CO 2 at 37 o C in a follicle dermal papilla cell growth medium kit (PromoCell) and subcultured when the cells reached 70 to 80% confluency. For experiments, the cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, USA) supplemented with 5% (v/v) fetal bovine serum (FBS; Sigma-Aldrich, USA). The human HaCaT keratinocytes (Thermo Fisher Scientific) were cultured in Epilife (Invitrogen, USA) supplemented with Human Keratinocytes Growth Supplement (HKGS; Invitrogen). 293T cells (American Type Culture Collection, USA) were cultured in DMEM supplemented with 10% (v/v) FBS. TCF/LEF luciferase reporter plasmids were obtained from Promega (USA). LY294002 was purchased from Merck (Germany).
3
Culturing Melanoma, Melanocyte, and Keratinocyte Cells
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B16F0 mouse melanoma cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Hyclone) and penicillin-streptomycin solution (Hyclone). Human epidermal melanocytes (HEM, Thermo Fisher Scientific, Waltham, MA, USA) were cultured in Medium 254 (Thermo Fisher Scientific) containing Human Melanocyte Growth Supplement (HMGS, Thermo Fisher Scientific). MNT1 human melanoma cells (ATCC) were cultured in MEM (Welgene, Korea) containing 10% DMEM, 20% FBS, and penicillin-streptomycin solution. HaCaT human keratinocytes (Thermo Fisher Scientific) were cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin. Human epidermal keratinocytes (HEK, Thermo Fisher Scientific) were cultured in Medium 254 (Thermo Fisher Scientific) containing Human Melanocyte Growth Supplement (HMGS, Thermo Fisher Scientific). These cells were incubated at 37 °C with 5% CO2.
4
In vitro Scratch Assay for Wound Healing
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The HaCaT human keratinocytes and HDFa human dermal fibroblasts were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dermal fibroblasts and keratinocytes were cultured using Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) contained with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco), 100 mg/mL of streptomycin (Gibco), and 100 mg/mL of penicillin (Gibco) at 37 °C in a humidified incubator with 5% (v/v) CO2. For in vitro scratch assay, human dermal fibroblasts or HaCaT keratinocytes were seeded in 12-well plates containing 10% FBS DMEM, triplicated at a density of 4 × 105 cells/well. After 24 h of the attachment period, the monolayers of cell surfaces were scratched with a sterile pipette tip and were incubated with a medium containing 5% FBS with or without KY19382 or PTD-DBM (positive control) dissolved in dimethyl sulfoxide (DMSO). After 24 h, cells were harvested, with a washing procedure with cold phosphate buffered saline (PBS, pH 7.4) carried out once, fixed in 4% paraformaldehyde (PFA) for 15 min at room temperature, and were stained with 2% (w/v) crystal violet. The wound closure area was measured using the NIS-Elements imaging software Ver. 4.50 (Nikon, Tokyo, Japan) (n = 3).
5
Culturing Human Cancer Cell Lines
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Three human cervical cancer cell lines, CaSki, SiHa and HeLa were purchased from American Type Culture Collection (ATCC). The CCCLs were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (CaSki) and Eagle's minimum essential medium (EMEM) (SiHa and HeLa). MCF-7 breast cancer cells, HFF-2 human foreskin fibroblasts (ATCC) and HaCaT human keratinocytes (Thermo Fisher Scientific) were used as control cell lines and were cultured in Dulbecco's Modified Eagle's Medium (DMEM). All three basal media were supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine, and 200 µg/ml penicillin-streptomycin. Cells were grown under sterile conditions in a humidified incubator at 37°C/5% CO₂.
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