Lionheart fx

After histochemical staining for the alkaline phosphatase activity and calcium deposition into the extracellular matrix by Alizarin Red staining, multi-well plates were allowed to air-dry and each well was scanned using high-resolution color brightfield objective (1.25X) of the Lionheart FX automated microscope (BioTek). For each scanned well, Image analysis was performed using Image J software according to the guidelines provided by the National Institute of Health. Briefly, images were converted to 8-bit grayscale and integrated pixel values were measured within a region of interest (ROI) covering entire culture area using same detection threshold for each multi-well plate. Data was combined from at least 3 different donor lines and represented as average fold change compared to control siRNA transfected cells without BMP2 stimulation.

After HUVECs were seeded onto 24-well plates and grown to 90% confluence, cell scratches were made with 200 μl pipette tips, and then PRP-Exos, PRP-AS and NC were added to the plates. The results were observed within 40 h by a Lionheart FX automated microscope (Biotek, USA).
HUVECs were seeded onto the upper chambers of transwell plates (3422, Corning) at a concentration of 4 x 10⁵/ml per 100 μl of the serum-free medium. The lower chambers were filled with 10% fetal bovine serum medium mixed with PRP-Exos, PRP-AS or NC. After incubation at 37°C for 16 h, the transwell plates were removed, fixed with 4% paraformaldehyde for 20 min and then stained with crystal violet dyeing solution for 15 min. The stained upper wells were wiped off with clean cotton swabs before photos were taken using a microscope system.

A total of 4000 U87MG cells or GSC5 cells/well were seeded in a 384-well low-attachment sphere plate (Corning, Lowell, MA, USA). The plates were then centrifuged at 300 g for 5 min. After 24 h, TAK901 and Matrigel were added to the spheres. After 24 h, the sphere area was calculated using a LionheartFX automated imager and Gen5 software (BioTek, Winooski, VT, USA).

The NAF-1^44–67 peptide was labeled with 5(6)-carboxyfluorescein at its N-termini as described in [15 (link)]. In a 96 well plate, 15,000 cells per well were allowed to grow for three days prior to experiments. Vesicles and PM were labeled with 5 µM of SynaptoRed^TM C2 in DMEM cell culture medium for 1 h and washed with DMEM cell culture media. The cells were then incubated in phenol red free DMEM with 100,000 U/ml Penicillin/Streptomycin with 15 µM of the peptide (Fl-NAF-1^44–67). The cells were imaged using a BioTek Lionheart FX (https://www.biotek.com) apparatus at 37 °C, 5% CO₂ under humidified condition using the following wavelengths 586/647 nm for the SynaptoRed^TM C2 and 469/525 nm for Fl-NAF-1^44–67. Image analysis and quantification were conducted using ImageJ (https://www.nih.gov). Briefly, the fluorescence signal of Fl- NAF-1^44–67 peptide was overlapped with phase contrast images and the intensity of signal was then discriminated between PM and intracellular localization.

EGF internalization was evaluated in HTR-8/SVneo cells using a modified EGF endocytosis assay [24 (link)]. After overnight serum starvation, cells were pre-treated with BPS (1 or 10 μg/mL) in 0.1% DMSO for 5 min, followed by a 5 min co-exposure with BPS (1 and 10 µg/mL) and Alexa Fluor Texas Red-conjugated EGF (100 ng/mL; biotinylated EGF complexed to Alexa Fluor 647 Streptavidin, Cat#: E13345, Thermo-Scientific, Philadelphia, PA, USA). The positive control group was exposed to 0.1% DMSO. Negative controls were treated with 100 ng/mL unlabeled recombinant human EGF (Cat#: E9644, I3390, MilliporeSigma, Saint Louis, MO, USA). Cells were then washed with pre-warmed PBS and incubated at 37 °C in serum-free IMD medium for 60 min, then fixed using a 1:1 methanol:acetone solution at −20 °C for 20 min, followed by three TBS washes. Cell nuclei were stained with DAPI (1:1000). For EGF endocytosis quantification, three random images (40X magnification) per well for a total of 3 wells per treatment group were obtained using (Lionheart FX, BioTek Instruments Inc, Winooski, VT, USA). Filter sets for 350 and 590 nm were used for detection of DAPI and Alexa Fluor Texas Red-conjugated EGF, respectively. Over 1800 cells from randomized fields from each group were analyzed using the software CellProfiler [67 (link)]. Total labeled EGF was normalized to the total number of nuclei.