Primary neurones culture - Survival problem primary neurones culture (Jun/15/2010 )

Hello,

I just start a PhD and I'm working on hippocampal adult neurogenesis. I have some survival troubles with my primary neurones culture for 3 months... The neurones look well till the forth day and then a lot of vacuoles appear in the soma. (Pictures attached) Somebody can help me?

I dissociate in a papaïne mix, then plate the cells in home pre-coated plate with poly-D-Lysine and borate buffer, left 1h30 in astrocyte medium for good adherence (neurobasal+FBS+P/S) then change all the medium for Neurobasal B27 conditioned overnight on astrocyte flask.

Thanks for you help!!!

-JulieS-

I make mouse hippocampal neuronal culture, which survives for 9 days. I didn't try to keep it longer, but you can try it. Do you want a procedure?

JulieS on Tue Jun 15 16:08:39 2010 said:

(Pictures attached) Somebody can help me?

I dissociate in a papaïne mix, then plate the cells in home pre-coated plate with poly-D-Lysine and borate buffer, left 1h30 in astrocyte medium for good adherence (neurobasal+FBS+P/S) then change all the medium for Neurobasal B27 conditioned overnight on astrocyte flask.

Thanks for you help!!!

-katenkak-

Hi katenkak,

Thanks for your answer! I would be interested trying your procedure!

-JulieS-

Hi!
I did not get any notifications about your reply, so I forgot to look up. I will write the procedure if you are still interested. I will check this page later.

-katenkak-

Procedure:
Pups 0-1 days old.
1. Cut heads of the pups, remove brains and dissect out hippocampi. Place them in a cold solution 1.
2. Chop with sterile scalpel blade first one way and thereafter again after turning the petri dish 90o.
3. Take 1 ml of solution 2 37oC (trypsin) add 600 µl to a new 10 ml tube and the rest to the cerebellum. Pipette the cells to the tube. Keep at room temp. Postnatal pups 4 days for 8 min and pups at 7-8 days for 14 min (10-11).
4. Add 3 ml solution 4 (mild trypsin Inhibitor).
5. Cfg. 1500 rpm 2 min.
6. Discard S/N. Fill up with 1 ml solution 3 (trypsin inhibitor) resuspend with the 1 ml blue tip (20 times) to make resuspension, carefully.
7. Leave the tube for 5 min in an angle position to allow remaining tissue blocks to settle. Transfer S/N with pipette to a new tube.
8. Add 3 ml of solution 5 and cfg. 5 min at 700 rpm.
9. Resuspend the cells in a suitable volume of medium neurobasal medium. Count cells.
10. Add 5microM cytosine arabinofuranoside after ca 48 hous to block mitosis of astrocytes by replacing ca half of the medium on neurons. Replace one third of the medium every ca third day and check that moisture level in the incubator is good enough to avoid evaporation of the medium.
Such neurons survived for 9 days-then I fixed them (may be they would survive longer).
Good luck!

MgSO4 stock solution, 150 mM:
3,7g MgSO4- 7(H20) in 100 ml sterile water. Sterile filter 0.2 µm
CaCl2 stock solution, 0,11 M:
0.162g CaCl-2(H20) in 10 ml sterile water. Sterile filter 0.2 µm.

Solutions: Sol 1: (ca. 20 ml/tube)
100 ml HBSS,
0.3 g bovine serum albumin (Sigma A4503),
0.8 ml 3.8% MgSO4-stock solution,
2 ml 1 M HEPES solution (Gibco 15630-056) to 20 mM.

Sol 2: (ca. 1.5 ml/tube)
12.5 ml sol 1
2.45 mg trypsin (Sigma T-0303, bovine pancreas, 15200 BAEE-U/mg prot).
Sterile filter 0.2 µm

Sol 3: (ca. 1.5 ml/tube)
10 ml sol 1
0.8 mg DNAse I (Sigma D5025, 1.5 Kunitz-U/mg solid)
5.2 mg Soybean Trypsin Inhibitor (Sigma T9128 1 mg inhibits 1.7 mg trypsin (10000BAEE-U/mg prot). Sterile filter 0.2 µm
100 µl l 3.8% MgSO4-stock sol.

Sol 4: (ca. 3.5 ml/tube)
10.5 ml sol 1
2 ml sol 3.

Sol 5: (ca. 3.5 ml/tube)
12.5 ml sol 1
15 µl from 1.2% CaCl2-stock sol (stock: 0.11 M CaCl2)
100 µl 3.8% MgSO4-stock sol (3.7 g MgSO4-7(H2O) in 100 ml water - 150 mM),Sterilefilter (Gelmann, 0.2 µm)
All solutions into 10 ml tubes, at –20oc

-katenkak-

Hi Julie,

I dont know if you are still checking this post..or if you will receive it on your email..but I am having troubles as well in developing neuronal cultures from adult mice. Did you solve the problem in the end?
my problem is that (i am doing hippocampal) i have a very low yeld, but anyways, when I plate the cells, the culture is very dirty, with a mix of round cells and grapes of 30-50 smaller cells..and i do no understand what i am seeing. I was wondering if you had this problem at the beguinning, and how the culture looks like to you when you first plate it.

do you have any picture of a neuronal culture of yours after a few hours from plating?

Marco

JulieS on Tue Jun 15 16:08:39 2010 said:

(Pictures attached) Somebody can help me?

-marcop-