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CALCULATIONS I

CALCULATIONS I. 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.

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CALCULATIONS I

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  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, dH2O • BSA for stability. Minimize sticking to vial. • Sterility is very important. • Minimize thawing-unthawing (degradation)

  3. Units You Should Know • 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)

  4. Tricks You Should Know • 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

  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. Example 1 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.

  7. Example 1 • 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

  8. Example 1 • 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

  9. Exercise 1 • 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

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

  11. Converting (mass/unit volume) concentrations to Molarities • 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 @ 10-6M • Factor X concentration is 2 mg/mL • Convert 2 mg/mL to Molarity • You need MW of factor X (10,000 g/mole)

  12. Converting (mass/unit volume) concentrations to Molarities • Use dilution formula to determine how much to use from stock solution • C1V1=C2V2 • Solve for V1=(C2V2)/C1 • V1=(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?

  13. Exercise 2 • 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

  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

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