Echo revolve microscope

To mimic the blastocyst-endometrial epithelium contact during attachment we co-cultured differentiated Sw71 spheroids with a confluent monolayer of HEC-1A cells for 24 h. The workflow included: (1) growing of the HEC‐1A cells in monolayer (≥ 95% confluency, 10 000 cells/well for 7 days) in McCoy's 5A complete in 96 well flat-bottomed plate (Costar) and (2) differentiation of Sw71 spheroids in DMEM-F12 complete in ULA plate (Costar) and (3) co-culture for 24 h. The day before spheroid transfer medium was switched to half McCoy’s 5A complete—half medium of interest (hsDMEM/F12, lsDMEM/F12 or Opti-MEM), each condition in triplicate). On the next day differentiated Sw71 spheroids were placed on the top of each HEC-1A monolayered well (4 per well) with wide-bore tips. After imaging the spheroids were left to adhere for 30 min, 1, 2, 4 and 24 h. At each time point the attachment rate (attached vs. placed spheroids) was determined after 3 min (min) shake at 1000 rpm or at 3000 rpm. The force of the shake of 1000 rpm corresponds to ~ 17 Hz frequency, while 3000 rpm—to 50 Hz frequency. Images were taken at each time point with ECHO Revolve microscope (RVL-100-M, Echo, San Diego, CA, USA).

The wound scratch assay was employed to evaluate the migration rates of cells cultured at different temperatures. First, cells were divided into three equal parts and placed into three 6-well cell culture plates, supplemented with GM, and cultured at a 25 °C incubator until they reached full confluency. A sterile 200 μL pipette tip was used to create a uniform scratch wound on the monolayer of cells, and then the cell debris was removed by washing with PBS 3 times. The scratch cells were cultured in serum-free medium at 21 °C, 25 °C, and 28 °C. Images were captured at 0, 12, and 24 h with an Echo Revolve microscope (ECHO, Chicago, America), and the scratch closure rate was measured using ImageJ software (v2.1.0, Bethesda, MD, USA).

Adipocyte size was measured using five images per animal on the Echo Revolve Microscope at 10× magnification (San Diego, California, USA). The image was divided into quartiles and a random number generator was used to select the quartile for quantification. The area of the quartile was measured through area tracing and the number of adipocytes counted for the given area. The average adipocyte size was determined by dividing the total number of adipocytes within the quartile by the quartile area.
For CLS determination, tissues were processed, embedded and F4/80 stained. Images were taken at 10× magnification. The image was divided into quartiles and a random number generator was used to select the quartile for CLS and area quantification. The average CLS was determined by dividing the total number of CLS within the quartile by the quartile area.

After animal sacrifice, mice were perfused with 10 cc of cold phosphate buffer saline through the left ventricle. The heart was transected across approximately the atrioventricular groove, and the ascending aorta, arch, and distal thoracic aorta were dissected from the mouse. The heart and aortic tissue were placed in 4% paraformaldehyde for 24 h at 4 °C and then transitioned to 70% ethanol. Fixed tissue was embedded in paraffin and sectioned at the aortic root for H + E, trichome, and elastin staining. Images were taken using the ECHO Revolve microscope (ECHO, San Diego, CA, USA). For elastin break quantification, four high power fields were captured from one 5 μm section across the aortic root at the level of the sinus of Valsalva. Elastin breaks were counted in each field within a 100 × 20 μm region of interest and averaged.

Images of immunostained catheters were obtained using the fluorescence ECHO-Revolve microscope (Echo). ImageJ (NIH image software) was used to quantify total cells as previously described [16 (link)], in areas of 0.45 mm² for the cell adhesion assay and 0.071 mm² for the catheter obstruction assay.