Expt 3 NO... -) (+) | 104% 1. Thaw the cell culture medium samples (both unstimu

Question

Expt 3 NO... -) (+) | 104% 1. Thaw the cell culture medium samples (both unstimulated and LPS-stimulated) prepared in the first lab period of this experimert n clean tubes, prepare 250 l each of 5. cell culture medium (not water) from the 1 M NaNO2 standard (each wii be a simple dilution rather than a serial dilution). BEFORE coming to class, calculate how you will prepare this set of standards. Record this in your notebook and on a separate sheet of paper to be handed in at the beginning of class, write out all of your calculations and include the followhg table showing what will be mixed with what to make each std (the table will be 10, 15, 20, 25, 30 and 40pM NaNO2 standards useful for prepping the standards) NaNo,standard volumell of NaNo,std Ivo ume lull culture medium 20 JM 25 uM 40 uM 3. Pipetting in the reverse mode (to avoid introducing air bubbles into the wells, which mess up the absorbance readings) add 50 /well of cell culture medium to wells 1A-D of the 96-well plate. The medium contains no nitrite and will serve as your blank. Pipetting in the 2 3

Explanation / Answer

This is can be calculated using a simple formula

V1C1=V2C2

Where V1= volume to be taken from stock, C1= stock concentration, V2=final volume, C2=required concentration

Now let's solve the problem

Stock concentration of NaNO2 = C1= 1 M

Volume OF 1M NaNO2 used for preparing working stock V2= X (We have to calculate)

Required volume of working stock = V2 = 250 microlitres

Now let's calculate the V1 for preparing 250 microlitres of 5 micromolar NaNO2

V1C1=V2C2

(X) x 1 = 250 x 40

X= 250 x 5 = 6250 picolitres = 0.00625 microlitres

So, in order to prepare 250 microlitres of 5 micromolar NaNO2, we have to take 0.00625 microlitres of 1 M NaNO2 i.e., from stock. Remaining we had to add culture medium. In this case it will be 250-0.00625 = 249.99375 microlitres.

Now for 10 micromolar NaNO2 it's very easy

5 micromolar = 0.00625 microlitres

10 micromolar =? (x)

x = (0.00625 x 10)/5 = 0.3125 microlitres of 1M NaNO2 and the rest is culture medium.

That is 250-0.3125=249.6875

Similarly we can calculate for the other concentrations.

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