Cytospin help

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MikeD520
MikeD520's picture
Cytospin help

I am growing cells in suspension and would like to do IF with them. Cytospin sounds like a good option, but I know nothing about it! Do I need a special centrifuge, or can I rig a swinging-bucket tabletop centrifuge to do it? I'm also not sure what speed to spin at, etc. Any help would be greatly appreciated!!!

Thanks,
Mike

Tracy
Tracy's picture
Cytospin is a relatively

Cytospin is a relatively straight-forward technique in which a single layer of cells is deposited onto a clearly-defined area of a glass slide. The slides are designed such that a "filter card" absorbs the excess fluid from the cells. This technique deposits the cells for very good nuclear presentation.

The most common machine is the Shandon Cytospin available from Thermo.

However there are some techniques out there to do it with a normal lab centrifuge like you suggest. This paper describes one if you have Science Direct Access. The protocol on IHC World with some nice pictures can be found here. (You have to link through our protocols section).

I also found this note of caution on the HistoListserv.

"Many years ago I tried cytospinning various types of cells,
cytospinning introduces so many of its own variables, such as rpm,
time of spinning and cell concentration, which markedly affected
the outcome of the quality of the cell prep, and the morphology
of the cell depending on its position on within the ring that I
abandoned it in favour of just smearing the cells
onto coated slides or made cell pellets embedded them in
agarose and treated it like a tissue block"
Good luck

 Tracy
Edited 03/10/09

AD
AD's picture
 Hi, 

 Hi, 
I am working on Plasmacytoid dendritic cells and as they are suspension cells, I am facing problems with attaching those on on glass slide for fluorescence microscopy...I have gone through the cytospin method, but is it absolutly necessary go by cytospin method or I can use normal smear method and then view under microscpoe? 
You said that cytospin is for nuclear presentation...so if I want to look at translocation of proteins into nucleus, will it be preferable to use cytospin method?
Please help me regarding this....

Thanks
Ashwini

samm
samm's picture
 If you want to quantitate

 If you want to quantitate protein translocation, you'll need to use cell fractionation to obtain cytoplasmic and nuclear fractions. Newer imaging flow cytometers such as the Amnis ISX is another option.
Cytospins or smears will just give you a qualitative picture.Use positively charged or lysine coated slides for pDCs.

AD
AD's picture
 Thanks samm...but I just

 Thanks samm...but I just want to check the presence of my desired protein into the pDC nucleus by fluorescence microscopy and I am using antibodies of flow cytometry...will it affect the viualization of fluorescence? because I am not ble to see any fluorescence under microscope...though I can see the fluorescence under flow cytometer...plz  help me...

Arpit Kakkar
Arpit Kakkar's picture
Find Lab centrifuge

I can help you to select lab centrifuge by giving more options of it so you can find according to your requirements and make your task easy.