Reagents, Solutions, and Media
1X PBS (phosphate buffered saline)
1.37 mM NaCl
2.7 mM KCl
4.3 mM Na2HPO4
1.47 mM KH2PO4
The pH should be 7.4 without adjustment. Bring to final volume of 100ml. Store at room
temperature.
10X HBSS Hank’s Buffered Salt Solution
NaCl (FW 58.44)
80.0g -137 mM
KCL (FW 74.55)
4.0g - 5.4 mM
MgCl2-6H2O (FW 203.3)
1.0g - 0.5 mM
MgSO4-7 H2O (FW 246.5) 1.0g - 0.4 mM
KH2PO4 (FW 136.1)
0.6g - 0.44 mM
Na2HPO4-7 H2O (FW268.1) 0.9g- 0.34 mM
For 1000 ml volume, use 850 mls (18Mohm H2O), add in order while stirring. Bring to
final volume of 1000mL in volumetric flask. Store at 4ºC.
1X HBSS Hank’s Buffered Salt Solution
-Mix 100 mls of the 10X HBSS solution (above) with 800 mls of 18Mohm H2O. Add the
following in order with stirring:
Anhydrous CaCl2 (FW111)
d-glucose (FW 180.2)
NaHCO3 (FW 84.01)
0.14g – 1.3 mM
1.00g - 5.5mM
0.35g - 4.2mM
-pH the solution to 7.4 and bring to 1 L volume in volumetric flask. Store for ~ 1 week at
4ºC.
50 mM KCl
NaCl (FW58.44)
KCL (FW 74.56)
MgCl2 (FW203.3)
CaCl2 (1 M stock
HEPES (FW 238.3)
Glucose (180.16)
5.0843g – 87mM
3.728g - 50mM
0.2033g – 1mM
5 ml 5mM Certified volumetric stock)
2.833g - ~12mM
1.8016g - 10mM
-1 L volume, pH 7.35, ~290-310 mOsm. Store at 4ºC.
1X Trypsin-EDTA Solution
Dilute trypsin (Invitrogen, Cat# 25200-056) in a calcium and magnesium-free solution
such as Hank’s Ca/Mg free saline (Invitrogen, Hank’s Balanced Salt Solution (HBSS)
(1X), Cat# 14170-120). Contains no calcium chloride, magnesium chloride, or
magnesium sulfate. Store at 4ºC.
Work quickly when using trypsin during cell culture , since trypsin degrades and
enzymatic activity declines at 37ºC.
Freezing Medium
Gibco Recovery Cell Culture Freezing Medium, Cat #12648-010
0.4% Trypan Blue
Dissolve 0.4g trypan blue in 100ml 1 x PBS or 1 x HBSS. Store at room temperature.
Dispase II
Roche, Cat #101658590001
Poly-L-lysine
Sigma, P1399
Complete Media
210ml RPMI 1640
25ml Horse Serum
12.5ml FBS
2.5ml P/S/G (100x)
250ml MPC Complete Media
420ml RPMI 1640
50ml Horse Serum
25ml FBS
5ml P/S/G (100x)
500ml MPC Complete Media
Place the following in water bath to thaw:
• Horse Serum (aliquots in -20 freezer)
• FBS (aliquots in -20 freezer)
• P/S/G (aliquots in -20 freezer)
When thawed place in hood with:
• 250 or 500ml Sterile Filter bottle (in tissue culture room)
• RPMI 1640 (tissue culture refrigerator)
To make solution:
 In a 500 ml sterile filter unit from Nalgene (blue top/PES filter membrane), add
volume of RPMI-1640, fetal bovine serum, horse serum, and 100x P/S/G
solutions.
 Filter with suction, remove the top and label appropriately. I mix only enough
media to use within a 2 week period because the L-glutamine will go off within
about 2 weeks.
 Store the complete media at 4 °C, and heat in a 37 °C water bath for ~1 hour prior
to use. Label bottle with Media type, date, and initials
 Feed cells every other day
Antibiotics: (P/S/G): Invitrogen Cat # 10378-016 (100X solution) Contains 10,000 units
of penicillin (base), 10,000 µg of streptomycin (base), and 29.2 mg of L-glutamine/ml in
0.85% saline, in a 10 mM citrate buffer to maintain penicillin potency.
Defined Equine Serum, Hyclone Cat # SH30074.03, triple 0.1 µm filtered, 500 ml. The
night before I aliquot, I transfer the bottle from the freezer to the refrigerator. Next
morning, I place the bottle, submerged with a weight to keep the media below the surface
of the water bath at 37 °C (but keeping the top of the screw-top lid well above the water
level). Once the serum is thawed completely, I increase the water bath temperature to 56
°C. Once the water bath has come to temperature, I let the serum heat inactivate for 30
min, mixing the bottle every 10 min or so to avoid gelling of the serum proteins. I aliquot
the horse serum into 25 ml aliquots stored in 50 ml Falcon conical tubes and freeze at -20
°C.
Defined fetal bovine serum, Hyclone Cat # SH30070.03, 40 nm filtered, 500 ml. I thaw
and aliquot the fetal bovine serum into 25 ml aliquots stored in 50 ml Falcon conical
tubes and freeze at -20 °C
We order fetal bovine sera (Cat #SH3007003) and horse sera (Cat #SH3007403) from
Hyclone through Fisher. The RPMI-1640 (Cat #SH3002702) media is also from
Hyclone.