Calcium Imaging of Organotypic Hippocampal Culture

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amye1083
amye1083's picture
Calcium Imaging of Organotypic Hippocampal Culture

I'm looking for either advice or maybe someone who has previously done calcium imaging with organotypic brain cultures. I'm looking to monitor the intracellular calcium levels while stimulating the culture or at least get an idea of how this stimulation changed the calcium levels from normal. I culture my brain slices on top of a millicell insert membrane. I have read that certain calcium-binding dyes have been known to attach to the membrane strongly, so I'm looking for possible dye suggestions and possible procedures for accomplishing intracellular calcium monitoring. Any help would be greatly appriciated!!!

Guy Sovak
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For the last couple of days I

For the last couple of days I tryed to find someone who could help with this Q but could not find any one that deals in this kind of research.
Maybe this link would help you:
http://www.bertholdtech.com/ww/en/pub/bioanalytik/applikation/camonitor.cfm
Never used or heard about this company or dyes

Guy

anurag pandey
anurag pandey's picture
amye1083 wrote:I'm looking

amye1083 wrote:

I'm looking for either advice or maybe someone who has previously done calcium imaging with organotypic brain cultures. I'm looking to monitor the intracellular calcium levels while stimulating the culture or at least get an idea of how this stimulation changed the calcium levels from normal. I culture my brain slices on top of a millicell insert membrane. I have read that certain calcium-binding dyes have been known to attach to the membrane strongly, so I'm looking for possible dye suggestions and possible procedures for accomplishing intracellular calcium monitoring. Any help would be greatly appriciated!!!

 
have you tried adding Oregon Green BAPTA 1 in patch pipette? it gets diffused in the cytoplasm within 15-20 min. depending upon quality of seal formed. you can also try Fluo-3 AM but problem with this is that it will bind unspecifically to any neuron. you can stimulate by using the same patch pipette if you are not stimulatin far from the cell.
 
 
 

dacrotty
dacrotty's picture
Protocol for Two-photon

Protocol for Two-photon calcium imaging of spines and dendrites in slices and neuronal cultures is available here:
http://cshprotocols.cshlp.org/cgi/content/abstract/2009/6/pdb.prot5231