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I have been trying to get my tumor cells to grow on gel microdroplets
with marginal success. But I am real good at making the droplets and my
cells love to be inside of them, which I do not want. Here is my protocol.
This is from a paper that I do not have at the moment; it is the work of
the people from One Cell Sciences. If you need the reference email me
and I will look harder for it.
Siliconize all glassware.
Combine 15-17mg agarose (Type IX, ultralow gelling temperature, Sigma
A5030) with 0.4 ml culture medium in a 15 ml polystyrene tissue culture
tube (Falcon #3033). Heat mixture for 4 min in a 90 degree water bath to
dissolve and melt the agarose, then cool for 4 minutes in a 37 degree
water bath. Add 0.1 ml of cells in culture medium. Add 5 ml autoclaved
mineral oil (USP type Borden and Remington) at 37 degrees (I have done
room temp also) and vortex vigorously for 15 seconds, dispersing the
aqueous suspension into liquid microdroplets surrounded by mineral oil.
An additional 5 ml of mineral oil is added and again vortexed for 15 seconds,
creating more microdroplets and also diluting them to reduce
their coalescence.
Chill the suspension for 20 minutes in an ice water bath, causing
gelation of the agarose and GMD formation. Pour this over 3 ml culture
medium in a 15 ml tube and centrifuge at 400g for 5 minutes, followed
by 800g for 5 min to transfer GMDs into the aqueous medium. Remove the
oil and any clumps of agarose and oil at the oi/water interface.
Finally, rinse GMDs with medium and pellet at 800 g for 4 min. This
yeilds GMDs suspended in the aqueous cell culture medium, cells inside
many of them, ready for incubation.
The GMD's are different sizes, but all pretty small; if I had been
more successful I had planned to simply filter out the big ones before
analaysis. The One Cell Science people claim that enough of whatever the
cells secrete is left inside the GMDs for analyis, but the droplets are
permeable to most small things, including antibodies....
Good luck
Deb Berglund
Montana State University
Microbiology
Bozeman, MT
On 18 Oct 1995, Mrs L M Dash wrote:
> I would like to select transfected cells on the basis of their secreted
> proteins. I have a paper (Kenney et al.) which describes the use of agarose
> microdrops and cell sorting to separate hybridomas producing appropriate
> antibodies.
> I would be grateful if anyone out there with experience with this technique
> has answers to the following questions:-
>
> 1) Do I need specialised eqipment to make reasonable microdrops? How much
> does it cost?
> 2) How straight forward is the method of preparation and sorting with
> microdrops? Are there any technical problems I can expect and do you have
> any tips for trouble shooting?
>
> Thanks in advance for your help,
> Laura Dash (Glaxo Wellcome).
>
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