Journal: Frontiers in Oncology

Article Title: A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

doi: 10.3389/fonc.2015.00177

Figure Lengend Snippet: Immortalized cells maintain morphologic characteristics of primary keratinocytes . Immortalized EGFR2 cells generated from an Egfr fl/fl mouse on an FVB/N background, immortalized B2F6 cells generated from an Erbb2 fl/fl mouse on an FVB/N background, immortalized B1B2F3 cells generated from an Egfr fl/fl /Erbb2 fl/fl mouse on an FVB/N background, immortalized Cdc25A fl/fl /Cre + (cell line 8b) keratinocytes on C57/Bl6 background, all at the indicated passage numbers (P), or primary keratinocytes from FVB/N mice were incubated in LoCa medium (left), or for 2 days in 0.25 mM calcium medium (middle), or HiCa medium (right). Phase contrast photos shown. Scale bar indicates 100 μm.

Article Snippet: High calcium medium (HiCa) described in Ref. , which is necessary for primary keratinocyte adherence to the plates, was prepared from Lonza BioWhittaker Minimum Essential Medium Eagle (EMEM), with Earle’s balanced salt solution, non-essential amino acids, and l -glutamine without calcium chloride (Walkersville, MD, USA) supplemented with 8% fetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA), 0.8% Pen Strep (Life Technologies, Grand Island, NY, USA), and 60 μM CaCl 2 .

Techniques: Generated, Incubation

Journal: Frontiers in Oncology

Article Title: A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

doi: 10.3389/fonc.2015.00177

Figure Lengend Snippet: Immortalized cells express keratinocyte markers and differentiate in response to increased calcium . Immortalized EGFR2 cells (P 42), immortalized B2F6 cells (P42), immortalized B1B2F3 cells (P29), immortalized 8b cells (P53), or primary keratinocytes from FVB/N mice were treated with LoCa or HiCa media for 48 h, and immuno fluorescence was performed with the indicated antibodies (green) (A–D) or without primary antibody (E) . DAPI indicates nuclei (blue). Scale bar indicates 20 μm.

Article Snippet: High calcium medium (HiCa) described in Ref. , which is necessary for primary keratinocyte adherence to the plates, was prepared from Lonza BioWhittaker Minimum Essential Medium Eagle (EMEM), with Earle’s balanced salt solution, non-essential amino acids, and l -glutamine without calcium chloride (Walkersville, MD, USA) supplemented with 8% fetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA), 0.8% Pen Strep (Life Technologies, Grand Island, NY, USA), and 60 μM CaCl 2 .

Techniques: Fluorescence

Journal: Frontiers in Oncology

Article Title: A Method for the Immortalization of Newborn Mouse Skin Keratinocytes

doi: 10.3389/fonc.2015.00177

Figure Lengend Snippet: Proliferation decreases in immortalized cells after serum deprivation or increased calcium . (A) 8b cells were cultured for 48 h in media with the indicated serum concentrations and propidium iodide flow cytometry performed to assess cell cycle phase. N = 1. Experiment is representative of several that were performed. (B) Immortalized EGFR2 cells (P42), immortalized B2F6 cells (P42), immortalized B1B2F3 cells (P29), immortalized 8b cells (P53), or primary keratinocytes from FVB/N mice were treated with LoCa or HiCa media for 48 h, and immunofluorescence was performed for proliferation marker Ki67 (green). DAPI indicates the nuclei (blue). Scale bar indicates 20 μm.

Article Snippet: High calcium medium (HiCa) described in Ref. , which is necessary for primary keratinocyte adherence to the plates, was prepared from Lonza BioWhittaker Minimum Essential Medium Eagle (EMEM), with Earle’s balanced salt solution, non-essential amino acids, and l -glutamine without calcium chloride (Walkersville, MD, USA) supplemented with 8% fetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA), 0.8% Pen Strep (Life Technologies, Grand Island, NY, USA), and 60 μM CaCl 2 .

Techniques: Cell Culture, Flow Cytometry, Immunofluorescence, Marker