Trypsin inhibitor

A modified Boyden chamber assay was performed by using 24-well microchemotaxis chambers (Corning Incorporated Life Sciences, Corning, NY, USA) (Fluoroblock^TM insert system). Human gingival fibroblasts were incubated with 4 μM Calcein AM solution (Dojindo) for 30 min at 37 °C. The cells were trypsinized, and the reaction was stopped by the addition of a trypsin inhibitor (Nacalai Tesque). The cells were washed with medium and resuspended in serum-free medium to a final concentration of approximately 2.5 × 10⁴ cells/500 μL.
The cell suspension was added to the upper chamber of a cell culture insert, and 750 µL of medium without (control) or with shikonin (0.01, 0.1, 1, 10, or 100 µM) was added to the lower chamber. Medium containing 10% FBS was added to the lower chamber as a positive control. The upper and lower wells were separated by a 3.0 µm pore size HTS Fluoroblock^TM Insert (Corning Incorporated Life Sciences). Cell migration was observed after 1, 3, and 5 h. The number of hGFs that passed through the filter to the lower chamber was evaluated by using SoftMax^® Pro Microplate Data Acquisition and Analysis software at 485/530 nm excitation/emission [47 (link)].

To prepare CD31⁺F4/80⁺ cells from E10.5 mouse brains, WT or Egfp Tg brains were mechanically triturated with a pipette in PBS on ice. To fractionate CD31⁺F4/80⁺NG2⁻ cells from yolk sacs, E10.5 R26-mCherry mouse yolk sacs were digested using Accutase (Nacalai Tesque) with 0.2% collagenase (Nacalai Tesque) and 0.005% trypsin inhibitor (Nacalai Tesque) for 1 hour at 37 °C then gently triturated with a pipette on ice. These cells were rinsed in PBS and then blocked with 3% normal mouse serum (Dako, Glostrup, Denmark) on ice for 10 min. Blocked cells were simultaneously stained with a PE-conjugated anti-F4/80 antibody (Bio-Rad Laboratories), an APC-conjugated anti-CD31 antibody (BD Biosciences) and, if needed, an Alexa488-conjugated anti-NG2 antibody (Merck Millipore) on ice for 15 min. Labeled cells were sorted using a FACSAria cell sorter (BD Biosciences). CD31⁺F4/80⁺ cells sorted from WT embryonic brains were seeded on fibronectin- or collagen type I-coated 8-well slides (BD Biosciences) and cultured in HuMedia-EG (KURABO, Osaka, Japan) for the durations indicated. CD31⁺F4/80⁺NG2⁻ cells sorted from R26-mCherry mouse yolk sacs were co-cultured with b.End5 cells and mouse neural stem/progenitor cells for the durations indicated.

We generated iHep cells from mouse dermal fibroblasts (MDFs) and conducted co-immunofluorescence staining of albumin with E-cadherin, as described previously (Sekiya and Suzuki, 2011 (link)). Some modifications that were added to the methods are specifically described below. In the preparation of MDFs, we incubated small pieces of skin tissue obtained from C57BL/6 adult mice (8–10 weeks of age) in Hanks' balanced salt solution containing 0.05% collagenase (Wako, Osaka, Japan) and 0.01% trypsin inhibitor (Nacalai Tesque, Kyoto, Japan) for 20 min at 37°C. The treated tissues were then collected by centrifugation (200 g for 3 min), plated on gelatin-coated six-well plates, and grown until expanded MDFs reached confluency. In the production of recombinant retroviruses, we used PLAT-E cells (Morita et al., 2000 (link)) for transfection of plasmid DNA. In this study, we used iHep cells at passage 9–12 for examination.