Diff quick solution

Cell migration was assessed using a 48-well Boyden microchemotaxis chamber (Neuro Probe Inc., Gaithersburg, MD, USA) as in a previous report [59 (link)]. Lower chambers were loaded with different concentrations of CSFAb or the positive control EGF (1 ng/mL) in DMEM containing 0.1% BSA. A membrane coated with type Ι collagen was laid over media in lower chambers. Upper chambers were loaded with HaCaT cells at a density of 5 × 10⁴ cells per well in DMEM supplemented with 0.1% BSA. Lower and upper chambers were then assembled and incubated at 37 °C for 4 h when the membrane was fixed and stained using Diff-Quick solution (Baxter Healthcare, Miami, FL, USA). Cells that migrated to the lower surface of the membrane were counted using an optical microscope at ×200. Levels of cell migration were presented as percentages of those of untreated controls.

A collagen sprout outgrowth assay was performed to simultaneously assess the migration and proliferation of HaCaT cells [27 (link)]. In brief, HaCaT cells (2.5 × 10⁷ cells/mL) were added to a solution of type I collagen, 10× DMEM, and 1N NaOH (pH 7.2). This mixture was spotted (2.5 × 10⁵ cells/10 μL) into the wells of a 24-well culture plate and dried. Spots were then incubated with various concentrations of CSFAb or the positive control (EGF, 50 ng/mL) at 37 °C for 48 h, and spots and sprouts were fixed and stained using Diff-Quick solution (Baxter Healthcare). Images of spots and sprouts were obtained using an optical microscope at ×100, and sprout lengths were analyzed using Scion Image software (Frederick, MD, USA). Sprout outgrowth levels are expressed as percentages of the sprout outgrowths of untreated controls.