Transcript CALCULATIONS I Reconstituting Cytokines/Growth Factors • Need To Supplement Cultures With Recombinant Growth factors/Cytokines • Issues To Consider – – – – – Recombinant factors are typically lyophilized (mg,

CALCULATIONS I
Reconstituting Cytokines/Growth Factors
• Need To Supplement Cultures With Recombinant
Growth factors/Cytokines
• Issues To Consider
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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)
Units You Should Know
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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)
Tricks You Should Know
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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
Formula For Reconstitution
M
C
V
C: concentration of factor (Ex. mg/mL)
M: mass of factor (Ex. mg, g, ng)
V: volume of reconstitution (Ex. mL, L)
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.
M
V
C
0.5g
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5g / mL
 0.1mL
 100 L
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
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
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
Exercise 1
• Volume: 100 L
• Aliquot: 2 L per tube (50 tubes)
• Avoid working with anything lower than 1
L, accuracy becomes unreliable.
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)
2  10 3 g 1 m ole 1000 m L
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 0.20 m M  200 M
1 mL
10,000 g 1 Liter
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?
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
Determining Approximate MW For
Proteins and Oligonucleotides
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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