Preparation of cytospin from single cell suspension.
Cell number, speed and trivial details affect cytospin .
Basic protocol:
Prepare a cell suspension of not more than 0.5 x 106 cells / ml of protein-containing medium.
Pre-label the slides.
Procedure for old type cytocentrifuge (be careful, this is not biohazard proof!!)
Prepare the slides mounted with the paper pad and the cuvette in the metal holder .
click to enlarge the image
Load up to 200 µl of this suspension in each cuvette.
Spin at 800 rpm for 3 min.
Extract the slide, paper and cuvette without disarranging.
Carefully detach the cuvette and the paper without damaging the fresh cytospin. Is very important to hold firmly together glass slide and cuvette when extracting from metal holder.
Mark the area around the cytocentrifuged cells with dry point or permanent marker.
Proceed with either immediate fixation or drying. Store unfixed cytospins for max 2 days at room temperature.
If you have little cells:
As few as 5 x 104 cells can be cytospun.
Proceed as follows:
Prepare the cell suspension with 10% FCS.
Pre run the loaded cytocentrifuge with 50 µl medium + serum, in order to wet the paper pads.
Run your sample with not fewer than 100 µl and not more than 250 µl.
Provider of cytospin material:
Shandon Inc. , 171 Industry Drive, Pittsburgh, PA 15275.
A: Shandon cards Cat n° 190005
B: re-used cuvette with glued paper residues removed.
C: Shandon pre-made cuvettes Cat n° 5991040 (box of 50).
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