I obtain few astrocytes after the subculture step.
Some astrocytes remain adhered to the culture dish surface after washing with PBS (Without Mg and Ca), incubating 15 min with PBS (Without Mg and Ca) and finally 10 min incubation with Trypsin-EDTA (0.25%).
I would like to be able to dissociate as many cells as possible without harming them.
The primary culture cells are seeded on a Tissue Culture Treated Corning® Petri dish. I subculture the cells a week after the primary culture.
Hello Alexander,
There are two answers to your question.
(1) If you want to lift and re-cultivate your 1o astrocytes, the Gibco's recombinant protease TrypLE is a safer alternative to trypsin. It is gentler on cells without sacrificing the efficacy. It is also stored at room temperature, and, unlike trypsin, does not loose its efficacy during freeze-thaw cycles.
(2) In our laboratory, we actually grow 1o astrocytes to confluency, lift them with TrypLE, freeze them in 90%FBS-10%DMSO, and store them up to 2 years. After re-plating these cells on poly-D-lysine containing dishes or T75, they re-grow without any issues and at high purity.
I hope it helps. If you need tips for how to grow high purity rat or mouse astrocytes, please let me know.
Good luck.
AM
Hello Alexander,
There are two answers to your question.
(1) If you want to lift and re-cultivate your 1o astrocytes, the Gibco's recombinant protease TrypLE is a safer alternative to trypsin. It is gentler on cells without sacrificing the efficacy. It is also stored at room temperature, and, unlike trypsin, does not loose its efficacy during freeze-thaw cycles.
(2) In our laboratory, we actually grow 1o astrocytes to confluency, lift them with TrypLE, freeze them in 90%FBS-10%DMSO, and store them up to 2 years. After re-plating these cells on poly-D-lysine containing dishes or T75, they re-grow without any issues and at high purity.
I hope it helps. If you need tips for how to grow high purity rat or mouse astrocytes, please let me know.
Good luck.
AM
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