Assemble the apparatus by following the diagram below:
The filter method (from Heraeus booklet)
A slide is put into the vessel holder (cytospin clip) and a suitable filter card is positioned between the container and the slide. Micro tubes with a hole in the bottom, which are placed into the container holes, are used to hold cell suspensions. Due to capillary forces, the sample only enters the container once the centrifugal force is applied. The suspended cells reach the glass surface and stick to it.The micro tubes prevent sample loss by stopping a flow on to the filter paper prior to centrifugation.
Depending on thesuspension's cell concentration, thesample quantity needs to vary. For this method the recommended sample volumes are between 50 to 200 pm.
The smaller the sample quantity the better it is absorbed by the filter paper. With larger volumes liquids collect in the bucket's cavity underneath the slide.
Method for Stromal Cell Keratin Staining (Dan Gray)
Sorted stromal cells were washed and cytospun onto slides (13300 cells per slide in 200uL of PBS) at 850gmax for 2 minutes.
Following air-drying, slides were fixed in methanol at 4oC (in fridge) for 40 minutes. This fixative is best for preserving kertains, others may be better for other proteins.
Cytospins were stained with rat anti-mouse K8 and rabbit anti-mouse K5 for 20 minutes, washed in PBS, then stained with Alexa-488 conjugated anti-rabbit IgG (Molecular Probes) and biotinylated anti-rat IgG2a (Southern Biotech), followed by Alexa-568 conjugated streptavidin (Molecular Probes).
Cytospins were also stained with 2.5mM TOTO (Molecular Probes) in PBS for 5 minutes, then washed and mounted.
Images were acquired on a Bio-Rad MRC 1024 confocal microscope with a three-line Kr/Ar laser (excitation lines 488, 568 and 647nm) using the acquisition software LaserSharp v 3.2 (Bio-Rad) and analysed using LaserSharp processing software.