Chicken eggs

Chicken eggs were purchased from Charles River Laboratories (Wilmington, MA, USA) and placed in an incubator at 37°C, 40% humidity. On day 3, up to 8 mL of albumin was aspirated from a small hole made at the bottom of the egg and the hole was sealed with candle wax. Then an approximately 2 cm large window was cracked into the rounded part of the upright egg using Dumont tweezers (6) and the egg membrane was completely removed. The window was covered with a cap of sterilized aluminium foil. The eggs were then incubated in a cell culture incubator at 37°C, 40% humidity, 3% CO₂. On day 7, up to 2 mm filter paper patches were punched out of sterilized Whatman-filter papers (Sigma Aldrich) and placed on a sterile surface. Five microlitres of treatment solution, control medium or ovarian cancer cell conditioned medium from OVCAR-3, OVCAR-5 or COV-362 cells, as indicated, were dropped on each filter paper allowing it to dry for 15 min and carefully placed on the developing CAM. Blood vessel development was observed daily and pictures were taken 3 days (day 10 of egg development) after treatment with a stereo microscope. Vessels crossing the outline of the filter paper were analysed using ImageJ (NIH). Six to nine independent experiments per group were performed in triplicate.

A/California/07/2009 (H1N1pdm09), A/Texas/50/2012 (H3N2), and B/Massachusetts/2/2012 viruses (International Reagent Resource; Manassas, VA) were amplified on fertilized chicken eggs (Charles River Laboratories, Wilmington, MA). Eggs were incubated at 37 °C with humidity and 0% CO₂ for 48 h, before transfer to 4 °C for an additional 24 h. Allantoic fluid was then collected, spun down to remove debris, and initially titered with turkey red blood cells (Lampire Biological Laboratories; Pipersville, PA). Of note, B/Massachusetts/2/2012 was not ether split prior to conducting serological assays.