Hello all

Recently, after we stained our cortical neuron culture with cytoplasmatic dye (ViaFluor or CFDA), we noticed that the culture is messy with debris, which are larger and much more fluorescent than the cells (see attachment. Blue is nuclei Hoechst staining, Green is CFDA/ViaFluor cytoplasmatic dye).

We use the same protocol to extract the cortical neuron for the last 2 years (described later), and the culture itself is growing fine with perfectly excitable neurons. We try to use a new stock of the dyes, repeat the experiment again, or extract new cortical neurons but without any success.

Can anyone help?

Thank you very much

Gal

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Briefly, the brains of 1d newborn rats are dissected, place in 15ml test tubes with PBS-TC (high glucose PBS), and incubated in an orbital shaker with Trypsin without EDTA for 15min at 37 degrees. The trypsinization is stopped by adding horse serum. After several minutes the medium from the test tube center is removed and additional PBS-TC and horse serum is added to complete 11ml. Then a vigorous mechanical pipetting is used in order to break the tissue and clusters and after 7 min of resting time (aims to separate the cells from the waste) the center of the test tube medium, which is supposed to have the cells, is transferred to a new sterile test tube, then after a more incubating time (aims to let the waste sink) the sank waste is removed and the cells are going to centrifugation (8.5min, 2800rpm). As the last step, the medium is replaced by a feeding medium (high glucose, B27, and Glutamine) and filtered by 20um filter to prevent clusters and tissue waste. The cells are incubated for 3-21 days, depends on the purpose of the experiment when replacing half of the medium every 3 days.