I would like some inputs regarding using the kind of culture dishes, media or any other requirements to start up live cell imaging to see ligand receptor interaction if any is happening in the membrane, or at least study the properties of my protein in a normal and mutant scenario.
In addition to the above, a heated stage is usually needed. If you're going to image over several hours, it's probably best if you can manage to supply an environment with CO2. Even for short-term though, remember that bicarbonate-buffered medium will lose CO2 within minutes and your pH will be off. I usually buffer with HEPES rather than bicarb for live cell imaging. The MatTek plates are great, but expensive. I found you can reuse them if you soak in 10% bleach for several hours, rinse very will with dH2O and then they can be gas sterilized. They used to offer a free sample sleeve so you can try them out.
As Pedro and Eric has suggested, environmental conditions are important. I was recently attending a vendor seminar and they have a variety of products for live cell culture and microscopic imaging. They are from IBIDI (http://ibidi.com/). Check out their website and you can see lots of useful info regarding cell culture and microscopic imaging. Take into consideration about the phototoxicity too. Good luck.
I would be using a Zeiss set up which is not a model for live cell imaging for more than 8 hrs but yes short term time lapses can be performed. It lacks a co2 chamber but my requirements will be less than half an hour.
Moreso, in subsequent experiments I do want to increase the chamber temperature beyond 37C, say 45C for visualizing temp effects on the protein.
I would like to know has anyone used atleast for a trial, plastic BD 35mm dishes? do they give lot of background ?
And for HEPES based buffering can I get a protocol as in what concentration of HEPES in final DMEM?
I did ask Mat tek guys for a sample but I am not able to find a distributor. Hope I get the samples.
We have protocols for live-cell imaging of membrane GPCRs using fluorescent ligands - I can provide more detail if you think it will be appropriate. You could start with
Methods in Molecular Biology, 2011, 746, 211-236
Detailed confocal imaging protocols on page 221.
Generally plastic dishes (other than Ibidi or other specialty dishes) are far too thick to allow any kind of proper resolution; you can also get a lot of backscatter/background from normal plastics. I have tried it, but with no useable results. Unless they are long working-distance objectives, most objectives are designed to see only through 0.17 mm or so, most dishes are around 0.8-1 mm thick. You may see fluorescence from your cells, but it's usually very difficult to make out any structures. You could have better luck if your objectives have a correction collar: http://www.nikoninstruments.com/Information-Center/Correction-Collar
I assume you are using an inverted scope, if you can find some very large coverslips you could try putting a cell suspension onto the coverglass for a couple trials. Though this would be very fragile and prone to breaking and dripping solution into your scope.
For short-term imaging I've usually used HBSS (our media all had phenol red), but the HEPES was 20 mM final concentration.
you are imaging from the bottom or the top? i'm usimg a ZEISS LSM 700 (objective from the top) and therefore i plate my cells (cardiomyocytes) on normal 2,5 cm plastic petri dishes (nunc), coated with matrigel. No matter if excited at 405, 488 or 555 nm i don't have problems wtih background, so far.
Hi, Jan if that is so then its a good thing. Ya we have Zeiss too and it is from top. But since I would be using adherent mammalian cell line, does a poly-lysine coat help or an uncoated dish or a feeder layer coated dish from BD which we routinely use for maintenance will do? Else il coat those dishes with poly-L lysine and use.
we also did experiments with poly d lysine coated dishes, which also worked quite well. A colleague of mine uses PDL coated coverslips to plate his cells (astrocytes) on it and those he will set into his measuring chamber. finally, in german we say: practice is superior to theory. good luck!
Kalpita, there is an alternative to CO2 supply if you cannot connect your microscope to a carbon-di-oxide cylinder. CO2independent medium is available with invitrogen. This reduces the need of CO2 supply during the live cell imaging. Addition of HEPES as suggested in earlier post also helps but not for long time. Good luck
Hi Rajesh, when you say HEPES not for long time, how long does it work out? an hour or less?
Can anyone suggest if during imaging the cells have to be in the media with serum or an external solution with HEPES and salts would suffice? Does the presence of bicarbonate based medium hinder imaging?
Hi Kalpita, In my experience 10mM HEPES was not be able to hold pH stable for more than 30 min. That is one reason I shifted to CO2 independent medium.
in our lab we use HEPES and/or CO2/HCO3- buffered solutions during imaging experimets. So, CO2 buffered solution must be gased with carbogen all the time (just hang a tube into your solution), but once you set the right pH (by adding the right amount of NaHCO3) it will be quite stabel. as Rajesh, i also had some problems with HEPES buffered solution but i just used to get bigger pH shift over night (especialy keeping in the fridge may cause some irriversible shift) , but during one day of measurements the pH did hardly change (even though i just have 5 mM HEPES). But you should take some time for setting pH, some times i did it when i was in a hurry and than i often got some drift.
Here are the solitions i use. You wont need that BDM and taurine, thats for heart cells but the rest is rather universal:
HEPES buffered solution
mM
NaCl 130
KCL 0.65
KH2PO4 1.2
MgSO4 3.2
Taurine 5
HEPES 5
Glucose 5.5
Saccharose 15
BDM 5
KOH 0.35
CO2/HCO3- buffered solution
mM
NaCl 104
KCL 1
KH2PO4 1.2
MgSO4 3.2
Taurine 5
HEPES 5
Glucose 5.5
Saccharose 15
BDM 5
NaHCO3 26
Hi Kalpita,
You can try the Live Cell Imgaing Solution from Invitrogen. You can find more on:
http://products.invitrogen.com/ivgn/product/A14291DJ
If you want, you can make your own!!! See attachment.
Hi Kalpita, did you try using the imaging solution suggested above? did it work?
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