We've been trying to run calcium imaging experiments with the red Calcium Crimson and somehow it does not load our neurons well. We are quite successful with Oregon Green BAPTA and Fluo4. These latter ones work well.
Now we need something that is red (possibly far red). We bought Calcium Crimson to use but we have lots of difficulties with that. Any ideas or tricks on how to use it?
I have worked with Ca crimson in vascular myocyte but the result is not beautiful. The most loading was obtained with 2mM crimson-AM, 30min, 37°C, in solution in M199 culture medium w/o phenol red. You can also try with ASANTE probe (in ratiometric mode or in non ratiometric mode in red). The probe is available via teflab or interchim.
Thanks Jean-Luc!
I think I'll try your 30 min loading protocol first. If it does not work still I can still try the rest.
Thanks a lot!
Best,
Bela
As red calcium indicators I used Fura Red (Invitrogen, Molecular Probes) that is excited at 420nm and emit at 650nm, Kd 140 nM. You can load the probe with adherent cells using 5uM of Fura Red in 1% BSA (bovine serum albumin) or 1% Pluronic F-127 in HBSS or HEPES physiological buffer, 30 min in incubator at 37°C.
It seems to work fine.
Hi Simone!
Thanks for the reply! Unfortunately I need to use higher excitation length light source (above 540 nm). The 420 is in the middle of the range that I want to avoid!
Thanks anyways!
best,
Bela
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