Cellmask orange plasma membrane stain

To assess cell membrane and total expression of HOMER3, immunocytochemistry assays were performed. HOMER3 expression was determined in methanol fixed cells using a rabbit polyclonal antibody (PA5–59383, Thermo Fisher Scientific), incubated 1:100 for 1 h at room temperature. A goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody Alexa Fluor 594 (1:1000, A-11012, Thermo Fisher Scientific) was then incubated for 30 min at room temperature prior to detection. For total HOMER3 detection cells were permeabilized with 0.2% Triton-X in PBS 1x for 5 min. Nuclei were stained using Vectashield mounting medium (Vector Laboratories) with DAPI. Membrane HOMER3 was also assessed according to the flow cytometry optimized protocol, using PI as cell death control and CellMask™ Orange plasma membrane stain (Invitrogen) to demark cells at 0.5x for 6 min at room temperature. For ST antigens detection, 4% PFA fixed cells were incubated with 5 μg/ml of fluorescein labeled PNA lectin (Vector Laboratories) for 1 h at room temperature after 4 h of α-neuraminidase from Clostridium perfringens (Sigma-Aldrich) digestion 0.1 U/mL at 37 °C. Immunofluorescence images were acquired using a Zeiss Axio Imager Z1 (Carl Zeiss) microscope through a Axiocam MR ver3.0 (Carl Zeiss) camera and using the Software Axiovision 4.8 (Carl Zeiss).

Ovarioles were dissected from ovaries of 7-day-old adult females and desheathed in medium containing 60% Schneider’s Drosophila medium and 40% Basic Medium Eagle as previously described [58 (link)], followed by incubation with fresh medium containing 0.1 μM JH III (Santa Cruz) for 1 h. In pharmacological experiments with inhibitors, ovarioles were separately exposed to ACPD (0.1 μM), ML141 (10 μM, MedChemExpress), suramin (1 μM, Sigma-Aldrich), genistein (10 μM, MedChemExpress) and Su6668 (10 μM, MedChemExpress) for 30 min, followed by JH III (Santa Cruz) treatment for additional 1 h. DMSO was used as the control. After the incubation, CellMask Orange Plasma Membrane Stain (Invitrogen) was added and further incubated for 5 min. The images were captured with a ZEISS LSM710 laser confocal microscope and processed with Zen2012 software (Carl Zeiss). Area of follicle cell and patency were measured with Image-Pro Plus 6.0 (NIH). Patency index was calculated by comparing the area of patency surrounding a follicle cell to the area of this follicle cell.

Chemically activated polyacrylamide gels on 18 mm coverslips (8kPa, Matrigen) were coated with 1 μg/mL fibronectin (Sigma Aldrich) before seeding HUVECs. HUVEC monolayer establishment, and activation were as described above. The day of the assay, a chemoattractant gradient was established by incubation of gels with 1 μg/mL SDF-1 for >2 h at 37 °C in a 5% CO₂ incubator. HUVEC monolayers were stained with CellMask Orange Plasma membrane stain (Invitrogen) before sorted shSdc1 or shCTRL bcCML cells were washed, resuspended in IMDM + 0.25% BSA, and added to the gels. Gels/cells were maintained at 37 °C and 5% CO₂ while imaging. Images were obtained using a Zeiss LSM-700 confocal microscope.

Particle diameter/size distribution and concentration in resulting EV preparations were analyzed by fNTA. For discrimination between biological and non-biological particles a fluorescent membrane dye was used. Analyses were performed on a ZetaView PMX110 instrument (Particle Metrix, Germany), and the corresponding software version 8.05.12 SP1 was used as described before [21 ]. In brief, EV preparations were stained with 5 µg/ml CellMask Orange Plasma Membrane Stain (Invitrogen, USA) and incubated for 30 minutes at 37 °C prior to appropriate sample dilution in 1x PBS. After instrument calibration, the temperature was clamped at 23 °C, and the pre-acquisition parameters to measure in the fluorescence mode were set to a shutter of 70, frame rate of 30 and a sensitivity of 95 % at high resolution. Post-acquisition parameters were as follows: minimum brightness of 25, size range of 5–1000 nm and a trace length of 15. Two cycles of measurement at eleven positions were conducted. To obtain EV concentrations of initial serum samples, a recalculation according to Eitan et al. [22 ] was applied accounting for sample dilution and EV sample and serum volume. Additionally, thereby obtained EV concentrations were normalized to the number of one million white blood cells.

WERI-Rb1 cells were stained with orange plasma membrane stain as per the manufacturer’s protocol (Cell Mask Orange Plasma membrane stain; cat#C10045, Invitrogen, Waltham, MA, USA) prior to the development of spheroids. The stained WERI-Rb1 cells were used for spheroid formation using the hanging drop protocol [29 (link)] on a low attachment U-bottom 96-well plate. Tight and regular spheroids (n = 9) were further exposed to IC₅₀ doses of topotecan, HC3 (IC₅₀ = 77.2 µL/mL), or HC5 (IC₅₀ = 65.2 µL/mL) for 48 h. The images were acquired using the automated high-content imaging system, IN Cell Analyzer 6000 (GE Healthcare, Singapore), equipped with a laser-based confocal platform having a 2× objective. The automatically captured single well imaging of spheroids and a Z stack of 11 images were separated by 50 µm total 500 µm depth for spheroids, using maximum intensity projection (MIP) acquisition and single-channel filter at 567 nm. To reduce data storage and image analysis time, we analyzed 2D projection images of tumor spheroids for multiple treatment conditions. Spheroid area and shape were automatically quantified by HCA using the IN-Cell 6000 software (GE Healthcare, Singapore). The mean area (µm²) of each spheroid was calculated to elucidate the drug/peptide response.