I am currently facing problems with my cell attachment and when the cells do not attach well they do not do well in the culture.
Hi Atossa, Thanks for the reply. Would you mind sharing your protocol for coating plates. Thanks,
Somasish
Fibronectin is from Gibco and the protocol comes with it. You just dilute it 1:50 in H2O
you cn also use matrigel and that is also from Gibco
Good luck
This is a good protocol http://www.jove.com/video/2056/how-to-culture-record-stimulate-neuronal-networks-on-micro-electrode
You can use glass coverslips by coating with 50 µg/ml poly-D-lysine (0.15 ml/cm2)(Sigma P6407). Incubate coated surfaces for at least 1 hour (up to 20). Aspirate the poly-D-lysine, rinse once with ddH2O, aspirate and air dry.
What I had done for years was to clean the coverslips before the start of neuronal cultures. Sonication helps when your cultures dont adhere properly onto the coverslips. If you dont have problem with adherence, then you dont need to worry about sonicating the coverslips.
In a 1-L beaker add enough 1 N nitric acid to cover the coverslips and sonicate for 1 h with the beaker covered using teflon wrap (with occasional swirling)
Then wash the coverslips three times with water (also with swirling). Don't swirl too hard, you might end up breaking a lot of coverslips.
Take caution while discarding nitric acid.
Then add enough 1 N HCl to cover the coverslips and sonicate for an hour with the beaker covered (swirl occasionally) .
Wash atleast 5 times with water and rinse 3 times with 95% ethanol and store the cs in 75% ethanol solution.
You can actually flame dry your coverslips before you start this whole process of coating.
Good luck.
I use 100ug/mL poly-l-lysine (Sigma catalog #P9155) in tissue culture water.
1. Add poly-l-lysine solution on the plates (volume depends on which plate you are using). Incubate the plates for 10-15 mins at RT. You may incubate at 37 C if needed.
2. Wash the pates with tissue culture water.
3. Let the plates dry for ~6 hrs in the tissue culture hood (you may even keep them overnight).
4. The plates are ready to use.
I used to use gelatiin-polylysine coating for culturing cortical neurons.: gelatin 0.08% in borate buffer pH 8.4, left in the dish overnight at 37, then wash with sterile distilled water, then polylysine 1mg/ml in borate buffer for 2-4h, wash twice with sterile water, suck off and let dry at 37 in humidified incubator. the dishes are ok for no longer than 7-10days.... it always worked very well for us. the polylysin solution can be saved and re-used up to four times.
You could indeed try fibronectin, I use it for mouse primary neural stem cells. Leave the coating for >2h.
Poly-L-lysine is often used as well.
i usually use the Poly-D-Lysine about 2-5 ml depend on the culture plate diameter and incubate it for the 2-3 hours and after incubation rinse the plate for 3 times with distilled water and dried in laminar flow hood air for few minutes.
Following Cel-Tak or Poly-lysine coating, plates can be air dried and stored at 4C for a week.
you can use poly-D-lysine (10mg/ml=stock concentration) diluted in PBS and incubate it for 30 minutes in room temperature. wash 3 times after 30 minutes with distilled water. i would like to share my protocol also..
Rose, your answer is a strong yes. Companies sell precoated plates. I would keep them at 20C. I would not personally advise saving plates that you coated yourself for more than a month.
you can use poly-D-lysine (10mg/ml=stock concentration) diluted in PBS and incubate it for 30 minutes in room temperature. Recently I am using Matrigel (1:1000), which is very good and support neurons.
Masuma Akter We use precoated cell culture plates for our primary neuronal culture. I still see neurospheres formation and aggregation of neuronal cells there. Do you recommend coating of Poly-L-Lysine or Poly-D-lysine on precoated plates for better neuronal cell attachment?
Rose O'Reilly Yes, you can store them, but not more than a month.
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