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No events in my EPSC


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#1 sharma

sharma

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Posted 21 December 2010 - 09:25 AM

Hello,
I am a graduate students who is trying to record spontaneous EPSCs from primary neuronal cultures obtained from chick ciliary ganglion (E8 i.e.e 8 days eggs post fertilization). These neurons are abundant in nicotinic receptors. my cultures look great but when i record using silgard coated pipettes i do not see events. Here's the protocol that i use. Any help whatsoever, would be deeply appreciated because i am in a very difficult situation.

E8 CG Culture

1. Acid wash the coverslips (Fisher, No 1, 12 mm) for 72 hours with Nitric Acid, then wash 20 times with Fisher boxed water, and twice with 95% ETOH (see seperate sheet), then dry. Put coverslips in the 24 well-dish before use.

2. Coat the coverslips with poly-L-Ornithine (Sigma, P-3655, 2 mg/ml stock stored at -20 C) (300 ul/coverslip (0.2-0.4 mg/ml), dissolved in 0.133M Borate Buffer, pH=8.4. The coating period has been suggested to be betwwen 2-24 hrs. P-DL-O (P 0671, Sigma) is used in Dr. Margiotta’s lab.

3. Wash coversliops with sterile Fisher Boxed water for at least 5 times, dry them and coat the coverslips with (300 l/coverslip, 10 - 40 g/ml in MEM) Laminin (Gibco, Cat#: 23017-015, stored at -80 C, ) for at least 1 hour(Again, it has been suggested to be between 2-24 hrs).

4. Wash the coverslips 3 times with MEM (Gibco), drain the MEM and put about 300 l culture medium (MEM, Glu, P/S, horse serum (Gibco)) to the wells, and put the plate in the CO2 incubator waiting for plating of cells.

5. Figure out the number of ganglia you need. For physiology, QNai use 2 ganglia/12 mm coverslip.

6. Dissect the ganglia under sterile condition, put them in sterile ice-cold Ca++ free Buffer eg. DF0 or MPG, in a 35-mm dish. Clean the nerve root and debris on CGs (as clean as possible). Then transfer the CG into another dish containing ice-cold DF0 or MPG. Make sure to pre-wet the transfer pipette, other wise the CG will stickto the glass wall. Cut CG in half or more times. Transfer these CGs into a clear tube containing ice-cold DF0 or MPG (Try to avoid debris in the Buffer. If you include debris in your culture, you will see quite a lot wierd noneuronal cells living happily together with your neurons. People using the cultures may not like to see that). Remove DF0 or MPG and add 1ml MEM++/hs/ 3% eye extract (MEM, Glu, P/S, hore serum and eye extract).

7. Pick a #9 pasteur glass pipette with smooth tip and flame the tip to ¼ or smaller of the original size. Pre-wet the pipette that you just made with culture medium for some times. Then trituate these CG until no big chunks are left (Trituate less than 20 times and avoid bubbles. Do not overtrituate the CG).

9. Add more culture medium to the volume you want. You may want to put 200 l medium including dispersed CG neurons to the 300 l medium already in wells.

10. The neuron might be evenly distributed in the well without extra treatment. If not, you may try to move your plate in a ‘8’style (like a bee, Ha).

11. Leave the plate in the hood for about 15 min to let the cells settle down, then transfer it the CO2 incubator.





Solutions used for E8 CG culture:

1. MEM with Earle’s salts (1x, Gibco, Cat. #: 11090), should be kept at 2 to 8 C, and protect from light.

2. Penicillin-streptomycin (Gibco, Cat#: 15140), should be stored at -5 to -20 C.

3. L-glutamine (Gibco, Cat#: 25030), kept at -5 to -20 C, and protect from light.

4. Horse Serum (hs, heat inactivated, Gibco, Cat#: 26050-088), kept at -5 to -20 C.

5. Eye extract, home made using E16-E18 embryoic chick eyes 1x MP buffer at 1 eye/ml. Make sure to remove cornea and lens and clean the muscle and other tissues attached to eye balls (as clean as possible). Grind for 2 x No. of eyes times using the glass homogenator. Better to do this on ice to keep eyes cold. Eye extract is good for at most 2 weeks
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MEM++hs
horse serum (v/v)
5% L-glutamine (v/v)
2.5% Penicillin-streptomycin (v/v)
(for now, QNai stores these liquid in freezer, and thaws them just before use, too cautious? Let’s try it in a different way later)
For 50 ml MEM++hs: 44.25 ml MEM (don’t pour everything to 500 ml MEM)
5.0 ml horse serum
0.50 ml L-glutamine (1st +)
0.25 ml penicillin-streptomycin (2nd +)

MEM++hs plus 3% eye extract: 48.5 ml MEM++hs
1.5 ml eye extract




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