hi

you can test Accutase or PBS + EDTA 0.2 % .

good luck

You may have to indicate where you want to isolate the cells. The technique to use will depend on the organ used to get the cells from. Organs with lots of connective tissue e.g lungs, require use of enzymes to free the cells, while organs with little connective tisssue e.g spleen will need mechanical disruption only. So you need to indicate the source of your cells

I want to prepare the single cell suspension from brain especially hippocampus to measure ADP/ATP Ratio.

For culturing neural stem cells from the SVZ, we dissociate the tissue with Accutase in addition to mechanical disruption (20min/37°C/1400rpm heating block). I assume dissociation of hippocampal tissue isn't that much of a difference. I have no experience with measuring the ADP/ATP ratio  though.

Best of luck!

I

If you want to cultured neurons the protocol may be as follow:

 Cell isolation and culture of cortical neurons:

Reagents:

- Minimum Essential Eagle’s Medium (EMEM) medium from (Bio-Whittaker). To 1000 ml of this medium we should be added  3.5 gs glucose  + 0.15 gs glutamine + 1 ml antibiotic mixture. Filtered with sterile filter on a sterile bottle an under sterile conditions.

- Antibiotic mixture: 1.2 gs peniciline + 2 gs estreptomicine + 0.4 gs gentamicine (You also may used a antibiotic mixture from GIBCO named Anti-Anti Reference 15240)

- Foetal calf serum. It is necessary for the culture because the cells die by apoptosis if you do not use serum. We may also use B27 suplemented medium in spite of serum. This is better.

- Locke without Ca2+ : NaCl (Pm = 58.44) = 8 gs + KCl (Pm =77.55) = 0.4 gs + Glucosa (Pm = 180) = 1 gs + HEPES (Pm = 238.3 = 1.192 gs + 1 ml of antibiotic mixture. Regulated to pH = 7,55 with NaOH. After that add NaHCO3 (Pm = 84.01) = 0.3 gs. Filtered though sterile filter on sterile bottle and in sterile conditions.

Cell isolation:

- Under sterile condition (sterile vitrine) make the follow:

- Foetal rat brains from rats at 19 days of gestation (E19) should be used. If you use adult rats may be you have not a good cell sepatation.

- Take away the meninges

- Place the brain on a Petry plate with EMEN medium

- Cut well with a small scissors

- Dispersed with and automatic pipette of 1 ml

- Filter by a BD Falcon sterile filter of 40 µm (Ref: 352340) and collect on a 50 ml centrifuge tube.

- Added Locke without Ca2+ untl 50 ml

- Centrifuged to 800 x g   during 15 m.

- Take away the supernatant

- Add Locke without Ca2+ to the precipitated (cells).

- Centrifuged to 900 x g during 15 m.

- Repeat this twice,

- Take away the supernatant and suspended the cells in medium EMEM +  1 ml of anti biotic mixture /100 ml and 1 ml of B-27 (or serum, take care with the serum, is better B-27)/ 100 ml

In this suspension are the cells. You should be counter to know the cell/ml.

If you want cultured the cells you should be prepared Petry plated with polilysine