Methods and Techniques Discussion => Flow Cytometry (FC) => Topic started by: fery on December 03, 2005, 12:53:23 PM

Title: preparation tissue for Flow cytometry analysis
Post by: fery on December 03, 2005, 12:53:23 PM
Dear Friends

please give me some directions about best protocol for preparation of solid tissue like skin for flow cytometry analysis.


Thanks in advance,


Fery
Title: preparation tissue for Flow cytometry analysis
Post by: excalibur on December 04, 2005, 02:43:26 PM
Michelle's fluid is used to ship tissues for flow to reference labs.
Title: Re: preparation tissue for Flow cytometry analysis
Post by: ImmunoNYC on December 04, 2005, 09:03:25 PM
Do you mean preservation like in your other post or actual disaggregation and preparation of whole skin for flow cytometry??

If you mean disaggregation you are likely going to have to collagenase and DNase treat the specimen for several hours to separate the cell populations. If you are just looking for expression of cell surface markers I think that immunofluorescence or immunohistochemistry may be a better way to go b/c of the tough nature of skin to separate and the possibility of not being able to account for all populations after separation. But this of course would depend on the end objective of your experiment.
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Quote from: "fery"
Dear Friends

please give me some directions about best protocol for preparation of solid tissue like skin for flow cytometry analysis.


Thanks in advance,


Fery
Title: preparation tissue for Flow cytometry analysis
Post by: fery on December 04, 2005, 11:16:37 PM
Dear Maximina


Because my previous experiments with IHC or IF wasn't succesful I have to try Flow cytometry.  I have some biopsy sample from skin and would like to detect my favorite cell in fresh tissue biopsy by Flow cytometry.
Because biopsy and flow analysis performe in different centers I also need a medium for handling and storage my biopsies for a short period of time .


Thanks in Advance


Fery
Title: preparation tissue for Flow cytometry analysis
Post by: excalibur on December 04, 2005, 11:45:49 PM
Michel's medium formula is on the Histology FAQs of this site.

http://www.ihcworld.com/_faq/fixation.htm#Michels
Title: preparation tissue for Flow cytometry analysis
Post by: ImmunoNYC on December 06, 2005, 12:47:44 AM
Excalibur detailed a medium for transport for you above as well as in several other posts. I suggest you check that out.

Also, for the flow cytometry I suggest you go to PubMed and look some references up and do exactly as they do. I do what you are asking weekly but not for skin (using collagenase, DNase and sometimes trypsin along with culture meshes and syringes - works well!). Skin is a potentially "tough" tissue to extract cells from so I wouldn't want to make guesses on what you should do. Look it up in published articles, it shouldn't be too hard to find. There may be particular enzymes you want to work with.

What antibodies are you using for immunostaining? Is your protein expressed on the surface? Perhaps we can try to work on why your staining didn't work also since that is our area of expertise :)


Quote from: "fery"
Dear Maximina


Because my previous experiments with IHC or IF wasn't succesful I have to try Flow cytometry.  I have some biopsy sample from skin and would like to detect my favorite cell in fresh tissue biopsy by Flow cytometry.
Because biopsy and flow analysis performe in different centers I also need a medium for handling and storage my biopsies for a short period of time .


Thanks in Advance


Fery