1. CALCULATIONS I

2. Reconstituting Cytokines/Growth Factors Need To Supplement Cultures With Recombinant Growth factors/Cytokines Issues To Consider –Recombinant factors are typically lyophilized (mg,  g) –Appropriate solvent Ex. PBS, dH 2 O –BSA for stability. Minimize sticking to vial. –Sterility is very important. –Minimize thawing-unthawing (degradation)

3. milli, Ex. mg (10 -3 ) micro, Ex.  L (10 -6 ) nano, Ex. ng (10 -9 ) pico, Ex. pg (10 -12 ) femto, Ex. fg (10 -15 ) Units You Should Know

4. 1 mg/mL SAME AS 1  g/  L 1 mg/mL SAME AS 1000  g/mL 1 mg/mL SAME AS 1 mg/1000  L 0.500 mg/mL SAME AS 500  g/mL 500  g/mL SAME AS 500 ng/  L Tricks You Should Know

5. Formula For Reconstitution C: concentration of factor (Ex. mg/mL) M: mass of factor (Ex. mg,  g, ng) V: volume of reconstitution (Ex. mL,  L)

6. A vial of GM-CSF of 0.5  g is available. The instructions state that the lowest concentration to use should be 5  g/mL. In addition they recommend to use PBS with BSA at a concentration of 500  g/mL. Example 1

7. Determine BSA mass (BSA is in powder form) You will need 100  L of solvent, which means 50  g. Can you weigh accurately 50  g? 0.000050 g!NO! Make 1,000 mL, This means 1000 mL x 500  g/mL 500,000  g, same as 500 mg, same as 0.5 g You can easily weigh 0.5 g Discard the rest Example 1

8. You determined 100  L volume How about aliquots, 1  L, 5  L, 20  L? Think of how you will use it. Let’s say you will be “feeding” a 10 mL culture at 10 ng/mL  10 mL x 10 ng/mL, 100 ng  20  L per tube (20  L x 5 ng/  L=100 ng) The objective is to avoid thawing/unthawing If you cannot avoid it, mark tube and use marked tube next time Example 1

9. You received a vial of rIL-2. Vial states 50  g Reconstitute @ 500 ng/  L Determine volume to reconstitute in and appropriate aliquoting if rIL-2 will be used to feed 100 mL cultures at 10 ng/mL Exercise 1

10. Volume: 100  L Aliquot: 2  L per tube (50 tubes) Avoid working with anything lower than 1  L, accuracy becomes unreliable. Exercise 1

11. Often you will know the Molar concentration to “feed” your culture and the factor concentration will be mass/volume Ex. “Feed” 10 mL culture with factor X @ 10 -6 M Factor X concentration is 2 mg/mL Convert 2 mg/mL to Molarity You need MW of factor X (10,000 g/mole) Converting (mass/unit volume) concentrations to Molarities

12. Use dilution formula to determine how much to use from stock solution C 1 V 1 =C 2 V 2 Solve for V 1 =(C 2 V 2 )/C 1 V 1 =(1  M x 10 mL) / 200  M =0.050 mL=50  L Note volume you are adding to culture has to be insignificant to culture volume for calculation to be accurate. Why? Converting (mass/unit volume) concentrations to Molarities

13. Stock concentration of cytokine: 2 mg/mL Determine Molarity (MW of cytokine: 200 KDa) Determine What Volume To Use To Feed 30 mL Of Culture at 10 -7 M Answer: 10  M; 300  L Exercise 2

14. Determining Approximate MW For Proteins and Oligonucleotides 125 g/mole for each amino acid  58 a/a cytokine (~125 g/mole)x(58) =7,250 g/mole OR 7.250 KDa Nucleic acids 325 g/mole  25 nucleotide oligo (325 g/mole x 25) =8,125 g/mole