Hi,
I am currently running an experiment that involves a 10-minute incubation of live cultured neurons in PBS containing magnesium and calcium on ice. Following immunocytochemistry for the neuronal marker MAP2, they appear either 'streaky' and inconsistent or blebby throughout the dish of cells.
The PBS-MC recipe I use is:
0.137 M NaCl
2.7 mM KCl
4.3 mM Na2HPO4.7H20
1.4 mM KH2PO4
1.33 mM MgCl2
0.133 mM CaCl2
I have tried adjusting the magnesium and calcium concentrations, have tested the osmolality as well as the addition of sucrose. I have also tested whether the ice is too cold, however, even room temperature gave the same results. I have also tried without magnesium and calcium. In separate experiments, I used aCSF with an osmolality of 290 mmol/kg, adjusting the PBS-MC recipe to be close to this value had no difference.
Any suggestions would be great
Thanks!
Dear Courteney!
Please You try following potocol:
Article Differential Labeling of Cell-surface and Internalized Prote...
1. Dissociated Hippocampal Neuron Culture
Cells may not be healthy in this culture condition. Were the healthy neurons shown here (one of the three pictures) cultured in the same condition you described here?
Chandra Somasundaram all these images are from the same cell preparation, the healthy image is from a dish that has just been treated and fixed (with 4% PFA) whereas the two unhealthy images are from a dish that has been incubated on ice in PBS-MC for 10 minutes. The incubation usually contains biotin, for the biotinylation of surface proteins, but I have not included it to save resources while I resolve the cell health issues.
I also check the health of the cells prior to treatment using phase contrast, where they look normal and healthy.
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