Tectal cell culture

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

2. Dissect brains in sterile HBST and collect them on ice
3. Transfer brains into sterile Ca/Mg free HBST+EDTA w/ fire-polished Pasteur pipette
4. Cut tecta into small pieces (100-200 um)
5. Rinse these pieces with cold Ca/Mg free HBST+EDTA buffer

(Can be skipped, if the solution looks clean)

6. Transfer all tissues into cold Ca/Mg free HBST+EDTA in eppendorff tube
7. Gently shake the tube in the cold room for 30 min
8. Centrifuge 800 rpm for 45 sec to collect the tissue
9. Resuspend in 0.1 mg/ml DNase contained Ca/Mg free HBST+EDTA
10. triturate with a fire-polished Pasteur pipettes
11. centrifuge to remove cellular debris
12. wash cells w/ large volume of Ca/Mg free HBST+EDTA
13. centrifuge to remove buffer
14. Resuspend cells in Culture medium
15. plate on poly-L-Lysine-coated 22mm coverslips
16. let cells settle for 1 hr
17. add media to around 1.0-1.5 ml
18. incubate cells at room temperature (18-24C) in humidified chamber
19. Take away half medium and add same amount of fresh medium every 2-3 days.

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