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

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

(Can be skipped, if the solution looks clean)

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

CaCl2Š2H2O

0.050

0.025

In ddH2O

246.48

0.83

MgSO4Š7H2O

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

CaCl2Š2H2O

0.088

 

3.9

MgSO4Š7H2O

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