Preparation of Solutions and Media
Neurology Lab
HBSS-A: for dissections
Hank’s BSS without Ca++ or Mg++ salts (from cellgro)
Remove 17.5 from 500ml bottle
Add HEPES 1M from UC Cell Culture at 1:100 dilution (5ml)
Add Penn/Strep from UC Cell Culture to final [] of 2.5X (12.5ml)
10% PMG: (“plating media for glia”) for plating astrocytes and Neurons.
use MEM
add 10% FBS (fetal bovine serum)
add 1% L-glutamine at a 1:100 dilution
add 1% Strep
10% PMG for refeeding (see feeding schedule for astrocytes)
use MEM
add 10% FBS
add 1% L-glutamine at a 1:100 dilution
add 100ul of Vitamin E and Sodium Selenite Solution (see below)
example: for 100ml of 10% PMG, add 10ml of FBS, and 1ml of L-Glutamine,
and 1% Streptomycin.
3%PMG with dBcAMP: for differentiating astrocytes when confluent
use MEM
add 3% FBS
add 1% L-Glutamine at a 1:100 dilution
add 100ul of Vitamin E and Sodium Selenite (see below)
add 1ml of dBcAMP stock solution
example: for 100ml of 3% PMG/dBcAMP, add 3ml of FBS, and 1ml of LGlutamine, and 100ul of Vit E/Na Selenite, and 1ml of dBcAMP and 1 ml
streptomycin. FILTER!
5% GCM: (“glial conditioned media) for feeding
Co-cultures
10 %PMG from confluent ASTROCYTE FLASKS that have been designated as
GCM flasks.
The media should have been exposed for at least 4 days and filter
5% GCM: for feeding co cultures
5% GCM is 10% GCM diluted 1:1 dilution with MEM.
IMPORTANT: The concentration of Glutamine in GCM is 0.1% at a 1:1000
dilution.
When feeding neurons, add media very slowly so that the neurons are not
washed off.
Neurobasal/ B27 supplement media for feeding Pure neurons
500 ml Neurobasal Medium
1% B27 supplement ( add 10 ml of 50 x B27 supplement)
0.1 % Glutamine ( 0.5 ml of 10 x Glutamax)
Papain and DNAse: for dissociating cells after dissection
Dissolve papain 40 units/ml in HBSS-A (1.9mg/ml)
Place in warm bath until dissolved
Divide into 2ml aliquots
Dissolve DNAse 1mg/ml in HBSS-A
Place in warm bath until dissolved
Divide into 4ml aliquots
For use, thaw and mix 2ml DNAse with 1ml Papain
A - Antibiotic strep
Cytosine Arabinoside Stock: for halting cell division when cell are confluent
Prepare a 200uM stock, filter, and store frozen in 50ml tubes.
24.3 mg in 500ml MEM= 200uM
Place container in a warm bath until completely dissolved before aliquoting
them into 50 ml tubes at 25 ml each.
dBcAMP stock
add 25 ml MEM to 250mg bottle and store frozen
200uM Vitamin E Phosphate and 100uM Sodium Selenite: for supplementing refeeding
media .
dissolve 5.55 mg Vitamin E Phosphate (tocopherol) in 5ml of glacial acetic acid
dissolve 8.65mg Sodium Selenite in 10ml sterile water for a stock solution of
5mM (50000x stock); store in refrigerator
add the 5ml Vitamin E/acetic acid solution + 44ml sterile water + 1ml of the 5mM
Sodium Selenite stock for the final volume of 50ml
store in refrigerator
Prepared by
Elizabeth Gum 1/24/06