Clsm 780

For immunocytochemistry, hCSCs were seeded in ibidi-μ-slides (ibidi GmbH, Gräfelfing, Germany) and treated as described above (Figure 1; Section 2.1). Biological replicates were performed of two individual serum donors. Cells were fixed in 4% paraformaldehyde (PFA), washed with phosphate-buffered saline (PBS) (Sigma Aldrich) and permeabilized in PBS with 0.02% Triton X-100 (Applichem, Darmstadt, Germany) supplemented with 5% goat serum for 30 min. The primary antibodies were diluted in PBS (mouse anti-Nestin 1:200 (Millipore, Burlington, MA, USA), rabbit anti-PhosphoHsp27 (Ser82) (Cell Signaling Technology, Danvers, MA, USA)) and applied for 1 h at room temperature (RT). After three washing steps, secondary fluorochrome-conjugated antibodies (Alexa 488 anti-mouse or Alexa 555 anti-rabbit, Invitrogen, Life Technologies GmbH, Carlsbad, CA, USA) were applied for 1 h at RT with a dilution ratio of 1:300. Nuclear staining was realized by incubation with 4,6-diamidin-2-phenylindol (DAPI) (1 μg/mL, Applichem) in PBS for 15 min at RT. Finally, the samples were mounted with Mowiol (self-made). Imaging was performed using a confocal laser scanning microscope (CLSM 780, Carl Zeiss, Oberkochen, Germany). Five images were taken of each treatment condition and serum-donor for following data analysis.

Cells were stained with blue fluorescent 4, 6-diamidino-2-phenyl-indole (DAPI, 10 μg/mL) in the absence of light at room temperature for 15 min. After that, cell pellets were collected, washed twice, and resuspended in PBS. Four milliliters of the DAPI-stained cells suspension were vigorously shaken with 1 mL of silicone oil, forming oil in water (o/w) emulsions. Spherical oil droplets from the o/w emulsion were transferred by using a micropipette to a slide glass and observed using confocal laser scanning microscopy (CLSM 780, Carl Zeiss, Oberlochen, Germany).

For observation of FITC-TP4 peptide in various sarkosyl buffers, 4 μg of TP4 peptides was dissolved in 20 μl sarkosyl buffer (0.25x, 0.5x and 1x Sar) and placed on glass slide for 30 min before examination. 1x Sar buffer contains 10 mM sodium phosphate, 100 mM NaCl, pH 7.4 and 2.6 mM sarkosyl (0.075% sarkosyl, w/v). Similarly, various concentrations of FITC-TP4 peptides (1, 4 and 16 μg/20 μl) or sarkosyl as indicated were also employed for the study. The morphologies and images of FITC-TP4 or Rhodamine-TP4 particles under various environments were observed and taken by CLSM 780 (ZEISS, Oberkochen, Germany).