Vitrogen Coating Flasks
1) Add 1 ml Vitrogen to 44 ml of cold 1X PBS.
2) Add 3 ml dilution for every 25 cm2 surface area of flask.
3) Tilt flask to make sure entire surface is coated
4) Store in air tight container, on even surface at 4oC for a minimum of overnight and a maximum of three weeks.
5) Only open airtight container under sterile hood.
Before plating cells into a Vitrogen coated flask
1) Aspirate Vitrogen mixture
2) Wash once with 10 ml DMEM/F12
3) Add warmed H14 medium.
If you coat the flask using vitrogen (collagen used to coat the flask) and leave the cells overnight (not 30 min, which some protocols state) in the fridge, the flask will be good for two months.
Do a 1:45 dilution. 1 ml collagen, ‘Purcol’ mixed with 44 ml of 1x PBS. Use 9 ml per T75 flask and put in fridge. Coat 5 of the tubes, then put it back in the bag that it came in to seal it, and leave them in the 4 C fridge.
You can use the collagen twice, using a little more collagen (i.e. 1.1 ml) so that you still have collagen remaining the second time again. Label the flasks with the date, otherwise you don’t know how old they are. Do not use the flasks after the second use.
Upon first taking the flask out from the fridge, wash the collagen and PBS away using plain T4 media (DMEM F12).
Use 3 ml to wash for T25, or 5 ml for T75. If the media you are using is very red, you know that the pH is high and the media is old- replace it soon.
Use 9 to 10 ml total volume media for T75, and put 750,000 cells in when splitting. Put 350,000 cells in the T25, the total volume should be 4-5 ml. Always use flasks with an air filter.