CBP Release Package — page 6

5) Spray gloved hands with 70% EtOH and rub hands together until EtOH has evaporated before opening incubator and handling cell culture flasks 6) Bring confluent T75 flask of Vero cells into the hood 7) Sterilize the exterior of PBS bottle by spraying with 70% EtOH and wiping with paper towels, Place in hood 8) Draw up media from T75 flask of cells with Pipet Aid and a 10m pipette. Draw from lower left corner of flask without disrupting the monolayer 9) Discard media in plastic waste container and pipette tip in the tip waste container 10)Unscrew cap of PBS bottle and let the cap sit loosely on top of the bottle 11) Use a Sml pipette and Pipet Aid to draw up 3ml of PBS. Use dominant hand to control Pipet Aid and the other to lift loosened PBS cap. 12) Place cap back on PBS bottle after drawing up desired volume. 13) Gently dispense 3ml of 1x PBS onto Vero cells by uncapping the flask and holding the flask at a 45° angle. Gently dispense the PBS by the top opening of the flask and let the PBS slowly run down the length of flask. 14) Cap the flask and rotate flask so PBS has covered monolayer of cells 15) Uncap flask and draw up the PBS with 5ml pipette. Draw from lower left corner of flask without disrupting the monolayer 16) Discard PBS in the plastic waste container. 17) Inoculate cells by adding 1m] of PEDv to cells. 18) Recap flask and place in incubator (37°C, 5% C02) for 1 hour 19)During incubation, place PEDv Maintenance Media in water bath 20) After 1 hour incubation, follow the same procedure in taking the flask out of the incubator and placing it in the laminar flow hood 21) Sterilize the exterior of PEDv Maintenance Media by spraying with 70% EtOH and wiping with paper towels. Place in hood. 22) Add 8ml of warm PEDv Maintenance Media to flask 23) Collect 200ul aliquot for PCR. Store at -20°C 24) Incubate at 37°C, 5% COz2 for 3 days. Virus induced cyotopathic effect should be visible under the microscope in Vero-76 by day 3 post infection 3 days later... 25) On day 3 post infection, freeze and thaw the flask 3 times at -20°C and room temperature, Follow instructions on Step 5 to take flask out of incubator 26) Sterilize laminar flow hood as instructed in Step 3 27) Sterilize Pipette Aid and conical holder/rack as instructed on Step 4 28) With a 10ml pipette and Pipette Aid, draw up everything in flask and transfer to a sterile 50ml conical 29) Centrifuge conical at 1500rpm for 10 minutes at room temperature 30) Collect supernatant and transfer to a new 50ml conical. Discard old conical with pellet 31) Collect 200ul aliquot for PCR