Axiocam icm1

For phase contrast microscopy 5 x 10⁶ events were sorted in 15 mL tubes coated with 5% BSA (Carl Roth, Germany). Sorted samples were transferred in 20 mL centrifugal concentrators of 30 kDa molecular cutoff and centrifuged at 3,400 x g for 5 min. Agarose pads were prepared on glass slides with 1% agarose standard (Carl Roth, Germany). Microscopy slides were prepared by pipetting 3 μL of the concentrated sample suspensions on top of the agarose pads placed on glass slides and covering with glass coverslips. Samples were observed with Axio Vert.A1 (Carl Zeiss) inverted microscope equipped with 100 x oil immersion objective and 10 x ocular magnification. Images were captured with AxioCam ICm1 (Carl Zeiss) camera using ZEN lite 2011 software (Carl Zeiss).

Zeiss spinning disk confocal microscope (Axio Observer Z1, ×40 objective) was used for time courses of membrane potential (Arclight) and intracellular [Ca⁺⁺] in H9c2 cells. For excitation, Yokogawa fibre was used at 488 nm, with emission collected at 506 nm.
Zeiss AxiovertC epifluorescent microscope (×40 objective) with a Zeiss AxioCam ICM1 was used for time courses of intramitochondrial [Ca⁺⁺] (CEPIA and GECO) and membrane potential (Arclight or DIBAC₄(3)) with excitation by a HBO50 mercury lamp with a 470 nm/515 nm Ex/Em filter block for green and a 546 nm/608 nm Ex/Em block for red fluorescence. Intensity was measured in ImageJ following background subtraction. For each measured field, one well, or one 20mm dish, was used. Each field was chosen at random. Visual fields measured 156μm×156μm (confocal) or 460μm×345μm (widefield). For each repeat, 2–5 fields were processed and the average normalised intensity measured (ImageJ). Data are presented as the mean and standard error of the mean (SEM) of at least 4 repeats.

Evaluation of the origin of the vacuoles observed in treated cells by phase contrast microscopy was carried out using various fluorescent probes, i.e., LysoTracker^®, ER-Tracker^® and MitoTracker^® (Molecular Probes, Life Technologies (Merelbeke, Belgium), as described by Colin et al. [121 (link)]. Briefly, U373 cells were seeded on glass coverslips placed in 6-well plates (Sarstedt) at least 24 h prior to the experiment to allow them to adhere and grow. Cells were then treated with the MeOH extract (70 μg/mL) or the EtOAc subfraction (40 μg/mL). During the last hour of treatment, the probes were added to the culture medium as follows: LysoTracker^® (75 nM), MitoTracker^® (300 nM) or ER-Tracker^® (0.5 µM). At the end of the incubation period, the coverslips were removed and washed in cold PBS before mounting them on microscope coverslips to take pictures of living cells with an Imager M2 fluorescence microscope coupled with the AxioCam ICm1 and AxioImager software (Carl Zeiss, Zaventem, Belgium).

Endothelial monolayer confluence on the HFM was assessed by 25 µM cell tracker green staining (Thermo Fisher Scientific, Dreieich, Germany) in serum-free EBM-2 for 45 min. In order to assess EC viability on AH and FN coated HFM, samples were stained for 30 min in EGM-2 containing the DNA-intercalating dye Höchst33342 (10 µg/mL) and 1 µg/mL calcein-am, or rather calcein red (Sigma-Aldrich, Taufkirchen, Germany), if ECs were already labeled with cell tracker green. Images were acquired using the Fluorescence-Stereomicroscope (StereoDiscovery V8, Zeiss, Jena, Germany) equipped with the camera (AxioCam ICm1, Zeiss, Germany).