DFs isolated from young LFD animals were cultured in monolayer condition or in coculture with keratinocytes nontransduced or transduced with Ad-Foxn1 or Ad-GFP (n = 5 per condition).
DF cell metabolic activity was measured using MTT colorimetric method as described previously [43 (link)]. Briefly, after 24 h and 48 h, sterile MTT solution (3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h. Next, the medium was removed and formazan crystals were dissolved in DMSO for 1 h. Absorbance was measured at 570 nm using a microplate reader (ASYS Hitech GmbH, UVM340, Biogenet; Biochrom Ltd., Cambridge, UK) and MicroWin 2000 software version V4.0 for Windows XP (Siemens, Monachium, Germany).
For the viability assay after 24 h of culture, cells were trypsinized, counted using the trypan blue exclusion method and resuspended in Annexin Binding Buffer (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) with addition of Annexin V Alexa Fluor 350 Conjugate (Life Technologies) and propidium iodide (Life Technologies). Samples were analyzed using flow cytometry with BD LSRFortessa and BD FACSDiva TM v6.2 software (Becton Dickinson, Franklin Lakes, NJ, USA).
DF cell metabolic activity was measured using MTT colorimetric method as described previously [43 (link)]. Briefly, after 24 h and 48 h, sterile MTT solution (3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for 4 h. Next, the medium was removed and formazan crystals were dissolved in DMSO for 1 h. Absorbance was measured at 570 nm using a microplate reader (ASYS Hitech GmbH, UVM340, Biogenet; Biochrom Ltd., Cambridge, UK) and MicroWin 2000 software version V4.0 for Windows XP (Siemens, Monachium, Germany).
For the viability assay after 24 h of culture, cells were trypsinized, counted using the trypan blue exclusion method and resuspended in Annexin Binding Buffer (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) with addition of Annexin V Alexa Fluor 350 Conjugate (Life Technologies) and propidium iodide (Life Technologies). Samples were analyzed using flow cytometry with BD LSRFortessa and BD FACSDiva TM v6.2 software (Becton Dickinson, Franklin Lakes, NJ, USA).
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