Question

You are setting up multiple lung cell incubations. These lung cells adhere to and grow on the bottom of a plastic well as a monolayer.

There is a 24-well plate. The culture medium, which comes pre-made from a commercial supplier) is added into the wells, onto the cells. Media supplements such as glucose, virus and drug are later added into the culture medium

In your experiment you will set up a 24-well plate, with 1 mL of culture medium in each of the wells. In the culture medium you will need to add glucose to a final concentration of 5 mM (to make the lung cells happy). 1,000 COVID-19 viruses will be added to 20 of the wells on each plate (this won’t make the cells so happy). The incubations will take place in the absence or presence of RDRPkill (concentrations ranging from 0 to 50 μM).

The glucose is supplied as a 500 mM stock solution. The virus is supplied as a suspension which contains 1 million viruses per μL. You have your RDRPkill stock solution which is 10 mM.

a. How would you set up ONE well containing 1 mL culture medium, 5 mM glucose, 1,000 COVID-19 viruses, and 50 μM RDRPkill?

b. When setting up the entire experiment, your supervisor suggests that you make up a Master Mix containing culture medium, virus and glucose – thus meaning that you only need add about 1 mL of this to each well How would you make up that master mix?

c. Explain, in general terms and perhaps using a picture of the plate or a table, how you would organise the plate.

molecular weight = 400

The image shows the 24 wells. Thats it. Nothing special

Answer 1

Thank you for the question. The answer is as follows:

For 24-well plate, you would need a mastermix of 25 mL (it would account for any pipetting error)

Since, you would or would not add RDRPkill, I would suggest you add 980 ul of mastermix and 20 ul of water or different concentration of RDRPkill.

A. Take 1 ul of glusose from stock of 500 mM to give 5 mM final molarity. Take 5 ul of RDRPkill from 10 mM stock to give final concentration of 50 uM final molarity.

Dilute the stock solution of covid cells by taking 0.1 ul of cells in 1 mL of dilution solution/media. Take 1 ul of covid cells from this media.

Finally have 1 ul of glucose, 5 ul of RDRPkill and 1 ul of covid cells. Add 993 ul of media to yield total 1 mL of media containing 5 mM Glucose, 50 uM and 1000 COVID cells.

b) If you want to have a master mix of glucose, media and covid, multiply the above calculation except RPRKill by 25 to yield 25 mL of mastermix. That is 25 ul of glucose, 25 ul of covid cells from the dilution prepared above and 950*25= 23.75 mL of media

c) The plate should be placed on the centre of biosafety cabinet in P3 or prefably P4 environment. All dilution should take place inside the environment. The addition of glucose and media and/or RPRkill should take place first. COVID dilution need to be last step in addition. All residual dilutions should be discarded by standard operating pocedure. Use a pipette to add the dilution of mastermix in the 24-Well plate. I am not sure whether u r allowing the growth of lung cell line before the addition of COVID or not and type of cell line use, however, you should allow the lung cell line to adhere to the media in the absence of COVID before trying your mixture.