I need to grow and maintain Salamander(Ambystoma tigrinum) Mueller cells for a week. I was wondering if anyone had experience culturing amphibian Glial cells and if they had any protocols or tips. Currently I can get a relatively (~60-70%) pure isolation, but 99% of them die within a 3 days. I currently grow them in Salamander Media: 108 mM NaCl, 2.5 mM KCl, 2 mM HEPES, 1 mM NaHCO3, 1.8 mM CaCl2, 0.5 mM NaH2PO4, 1 mM NaHCO3, 24 mM glucose, 0.5 mM MgCl2, 1 mM Na pyruvate, 7% medium 199, 1x minimum essential (MEM) vitamin mix, 0.1x MEM essential amino acids, 0.1x MEM nonessential amino acids, 2 mM glutamine, 2 µg/mL bovine insulin, 1 µg/mL transferrin, 5 mM taurine, 0.8 µg/mL thyroxin, 10 µg/mL gentamicin, and 1 mg/mL bovine serum albumin (pH 7.7). Cultures are maintained at 10°C. I have also tried: increasing K+ to 15 mM, decreasing glucose to 0.5 mM, adding 10% FBS, changing O2 levels by increasing and decreasing amount of culture media.
Thanks for any help.
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Salamander Mueller Glia
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