Bovine Pituitary Extract
Q1. You wish to prepare a total volume of the 500 ml of the Keratinocyte Growth Medium. Calculate the missing values to complete Table 1.
(a) What will be the final concentration of Bovine Pituitary Extract?
(b) What volume of Epidermal Growth Factor stock do you need to add?
(c) What is the concentration of the Epinephrine stock?
(d) What volume of CaCl2 stock do you need to add?
Removing the cells from the dish using trypsinisation
Once the cells are sufficiently grown (confluent), they need to be removed from the culture dish and counted in preparation for the cell migration experiments. The liquid medium is removed from the dish and the cells are washed in pre-ZaUPed (37ÛC) 1[ ShRVphate buffered saline (PBS). 1ml of 1x Trypsin-EDTA iQ PBS iV added WR Whe ceOOV aQd Whe\ aUe iQcXbaWed fRU 2 PiQXWeV aW 37ÛC WR detach the cells from the dish. The trypsin is then deactivated by further addition of 5% foetal
bovine serum (FBS) in PBS.
Q2. PBS fRU Whe e[SeUiPeQWV (³1[ VROXWiRQ´) cRQWaiQV:
NaCl (Mr = 58.44) 137 mmol/l
KCl (Mr = 74.55) 2.7 mmol/l
Na2HPO4 (Mr = 141.96) 10 mmol/l
KH2PO4 (Mr = 136.09) 1.8 mmol/l
To facilitate these experiments you make up a 1 litre stock of 10x PBS.
(a) How much NaCl will you need to add to this 10x PBS stock
(b) How much KCl will you need to add?
(c) How much Na2HPO4 will you need to add?
(d) How much KH2PO4 will you need to add?
(e) The 10X PBS solution needs to be adjusted to a final pH of 7.4. In practical terms, how would you make up this solution?
Q3. The Trypsin-EDTA solution used in the experiment would contain 0.25% trypsin (w/v) and 0.02% EDTA (w/v). It is provided to you as a 10x stock solution.
(a) EDTA has a molecular weight of 292.24. What is the molarity of the EDTA in the 10X stock solution?
(b) What is the total final amount (in mg) of Trypsin which is added to the cells?
Q4. Following a 2 minute incubation, FBS solution is then added to the cells to deactivate thetrypsin enzyme. The solution comprises 5% FBS, made up in 1x PBS. You decide to make up 150
ml of this solution using your 10 x PBS stock, and a 100% FBS stock.
(a) How much 10 x PBS will you need to use to make the 150 ml solution?
(b) How much 100% stock FBS would you need? (2 marks )