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Dear Colin
The thermo-reversible live cell mountants CyGEL / CyGEL Sustain are designed exactly for this purpose. Immobilizing your cells in this hydrogel allows inversion.
I have been using coverglass petri-dishes for the imaging of drosophila embryos and zebrafish in addition to eukaryotic and prokaryotic cells, tumor spheroids and tissue pieces to keep these objects immobilized for high-resolution microscopy. This can also be done with coverglass-bottomed chambered slides and standard slides with a coverslip. You will be familiar with CyGEL's use for C elegans immobilization.
For the petri-dish approach, a silicone o-ring/washer (ca. 8mm i.d., 1.5 mm deep) is smeared with silicone grease and bedded down onto the coverglass for a waterproof seal and a "well" is created with an approximate volume of 100ul. Adherent cells can be added and allowed to adhere, and all but ca. 10% of the medium removed and replaced with CyGEL Sustain pre-doped with 10X culture medium. CyGEL Sustain is added cool (<18degC) and allowed to warm, setting and therefore immobilizing your cells for imaging. Cells should be good this way for at least 2 hours and can be recovered for orthogonal molecular analysis, flow cytometry or correlative microscopy techniques. Recovery is achieved by simply re-cooling the slide to liquefy the CyGEL, dilute and wash with cooled culture medium.
For non-adherent cells, these can be admixed to the cooled CyGEL Sustain (doped with CM of choice) and pipetted into the well. If required, a chambered slide can be cold-centrifuged to get cells into a narrow optical plane or simply allowed to settle by keeping the CyGEL cool for this purpose. Large objects can be placed into the CyGEL mountant directly, and positioned as required with a gel-loader tip.
If necessary, to limit dehydration, a coverslip can be overlaid on top of the o-ring "well" or alternatively cover the CyGEL surface with immersion oil - these are immiscible.
CyGEL/CyGEL Sustain are optically clear and inert, with RI almost identical to water and have negligible visible range fluorescence. They can be doped with nuclear counterstains (e.g. DRAQ5, for live cells) and viability dyes (e.g. DRAQ7) and other small molecules such as functional probes for lysosomes/mitochondria and anaesthetic, for the more vigorously motile subjects!
You can find a review describing this technology with application examples and recent publications in the July / August issue of American Biotechnology Laboratory (
http://www.nxtbook.com/nxtbooks/isc/abl_20100708/#/14)
or link to a download pdf version (
http://www.biostatus.com/resources/technical_documents/cygel_review_american_biotechnology_laboratory/).
You can, of course, contact me off-line for further discussions.
Very best regards
Roy
Roy Edward
Biostatus Ltd.
Tel: +44 1509 558 163 Fax: +44 1509 651 061
[hidden email] www.biostatus.com
Company No: 3079239. Reg'd in England and Wales.
Reg'd office: Biostatus Ltd, 56 Charnwood Road, Shepshed, LEICS. LE12 9NP, UK.
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