Bimane Media (hRPMI) Formulation Protocol
Contact: Ometa Herman
ometa@stanford.edu
|
Materials |
Qty |
Location |
Order Info |
|
RPMI Medium 1640 (w/o NaHCO3, w/ HEPES) |
1x10L |
B-008 cold room |
GibcoBRL 23400-062 |
|
NaCl sol'n |
1M |
|
|
|
Conductivity meter |
|
B-013 next to pH meter |
Fisher brand |
|
Oakton 12,880 uS Standard solution |
|
B-013 |
WD-00606-10 |
|
Millipak-40 0.22um disposable filter |
|
B-011 near sink |
Millipore MPGL04GH2 |
Notes:
·
1 M solution of
non-dissociable solute = 1 osmolar (Os)
·
1 M solution of
disociable solute = n Os, where n is the number of ions produced per molecule
of solute
·
Blood plasma = 0.308
Os
·
0.308 Os NaCl =
0.154 M NaCl
Methods:
Refer to
the Conductivity
Meter Operation protocol
for detailed instructions on its operation.
1.
Mix 1 bottle of RPMI
1640 with 10L ddH2O in a carboy.
2.
Stir until powder
goes into solution.
3.
Measure osmolarity
using the conductivity meter.
4.
The target
conductivity is 14,940 uS (=265 mOs KCl).
Add 1M NaCl to the RPMI until it meets the target.
5.
The volume of 1M
NaCl required can be estimated using the following formula:
Vs
= Vr (C - C0) / (2 - C)
Where
Vs = required volume of 1M NaCl solution
Vr = volume of RPMI (10L)
C0 = starting osmolarity (KCl equiv)
C = final desired osmolarity (KCl equiv)
6.
In addition, the
following conversion factors may be useful:
ppm = uS x 0.66
mOs
KCl equiv = ppm x 200 mOs / 7447 ppm
7.
Adjust to pH 7.4
8.
Fine tune
conductivity to meet target.
9.
Filter the 10L into
200 ml aliquots in TC flasks using a peristaltic pump and a Millipak-40 filter.