Question

Helpppp

Your cells are ready to split and you prepare to scale-up. Write a protocol for this scale-up in enough detail that anyone in your laboratory could follow your exact procedure. Your protocol should answer the following questions. How will you release the cells from the substrate? How will you split the contents of the flask (what kind of flasks, how many flasks, how much medium, etc.) for scale-up (10 points)? All of your flasks of cells are ready to be frozen back. Write a detailed protocol for freezing back your cells. Your protocal should answer the following questions (you can begin after trypsinization). At what concentration will you be freezing back your cells? What cryoprotectant will you be using? How much and how did you prepare the cryoprotectant media? Provide a fake cell count (from the hemocytometer), and assuming that all of your flasks have the same cell concentration and cell number, describe how you will get your cells from the tubes to the freezing vials (at the cell concentration that you mentioned) (10 points).

Answer 1

• Take the cell culture flask whose cells are need to be splitted.
• Aspirate out all the culture medium carefully by tilting up the flask. Add 1ml of Trypsin EDTA (0.05%) to the cell monolayer and keep it for 2-3 minutes at 37⁰C. (Trypsin EDTA is used to detach the cells from the culture flask)
• Add 10 ml of culture medium to the flask and collect the detached cells into a centrifuge tube. pellet down the cells by spinning the cell suspension at 300Xg for 5-10 min. Discard the upper culture medium and add about 1 ml of culture medium to the cell pellet. gently resuspend the cells and seed them into a T 75 flask and add 20-30 ml of culture medium.
• If the initial cell number is high, seed the cells (after detachment) into two T75 flasks with equal amount of media.
• Keep the culture flasks at the CO₂ incubator.