Tectal cell culture
12/20/2003
Coverslip preparation:
Clean up coverslips:
Immerse coverslips in 100% EtOH for 20 min
Rinse in d.d. H2O for 3 times
Imerse coverslip in d.d. H2O for overnight
Autoclave
Dry
Poly-L-Lysine solution:
Filter-sterilize 0.1M broic acid (pH=8.3 w/ NaOH)
Dilute Poly-L-Lysine (50X) in boric acid to the final concentration of 50 μg/ml
Coat coverslips:
Lay out coverslips in small Petri-dishs
Place 50 μl Poly-L-Lysine in the center of the coverslip
Push down on coverslip with pipette tip as you add solution to make sure it is flat
Let air dry for 1 hr
Rinse the coverslip with sterilized H2O
Air dry
These coverslips can be stored at room temperature for more than one month.
Tectal cell culture
1. Anesthetized tadpoles in Filter-sterilized 0.1% MS222
(Can be skipped, if the solution looks clean)
Buffers and media
HBST: Hepes buffered Steinberg’s Solution (from Saul Zakson)
Note: make all solutions in glass distilled water. Use plasticware or glassware washed with EtOH. Sterilize all solutions with 0.2 μm filters.
HBST:
MW |
Conc (mM) |
Salt |
per Liter (g) |
per 500 mL (g) |
|
58.44 |
58.18 |
NaCl |
3.400 |
1.700 |
|
74.56 |
0.67 |
KCl |
0.050 |
0.025 |
|
147.02 |
0.34 |
CaCl2·2H2O |
0.050 |
0.025 |
In ddH2O |
246.48 |
0.83 |
MgSO4·7H2O |
0.205 |
0.103 |
Adjust pH to 7.4 |
238.31 |
4.62 |
HEPES |
1.100 |
0.550 |
( ~1ml 1N NaOH) |
HBST-EDTA:
MW |
Conc (mM) |
Salt |
per Liter (g) |
per 500 mL (g) |
|
58.44 |
58.18 |
NaCl |
3.400 |
1.700 |
|
74.56 |
0.67 |
KCl |
0.050 |
0.025 |
|
238.31 |
4.62 |
HEPES |
1.100 |
0.550 |
In ddH2O |
372.24 |
0.4 |
EDTA |
0.149 |
0.075 |
Adjust pH to 7.4 |
|
|
|
|
|
(~1ml 1N NaOH) |
MEDIA:
5 parts: |
L-15 |
125 mL |
4 parts: |
buffer |
100 mL |
1 part: |
serum-plus |
25 mL |
BUFFER :
Final Conc (mM) |
|
per Liter (g) |
|
95.0 |
NaCl |
5.550 |
|
1.0 |
KCl |
0.075 |
|
0.6 |
CaCl2·2H2O |
0.088 |
|
3.9 |
MgSO4·7H2O |
0.961 |
In ddH2O |
9.4 |
HEPES |
2.240 |
Adjust pH to 7.4 |
Serum-plus :
Final Concentration |
Stock solution Concentration |
Final Concentration |
Amt of stock added to 25 mL of bovine serum |
|
Sodium selenite |
2.5 μg/ml |
5 ng/ml |
50 ul |
|
Transferrin |
2.5 mg/ml |
5 μg/ml |
50 ul |
|
Insulin |
2.5 mg/ml |
5 μg/ml |
50 ul |
Adjust pH to 7.4 |
* Add penicillin/Streptavidin/Glutamine (100X from Pranav) into the final mixture of the medium