Do Collagenases cause receptors to cleave off?

Use this category for questions regarding various immunological methods

Moderator: mdfenko

Do Collagenases cause receptors to cleave off?

Postby Whelans2 » Jun 08 2016 2:01 pm

I am really interested in a novel receptor on T cells, but the expression of it is really low on tumours. I am just wondering is it possible that different collagenases are cleaving off the receptor during processing. I was thinking of doing an experiment with different collagenase cocktails.
Whelans2
technophile
technophile
 
Posts: 14
Joined: Jun 08 2016 1:50 pm

Re: Do Collagenases cause receptors to cleave off?

Postby relaxin » Jun 10 2016 11:15 am

Some of the collagenases used in cell isolation are very crude, and hence may contain proteinases. The contaminating proteinases may cleave off cell surface receptors. You may need to buy collagenase of high purity.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7185
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: Do Collagenases cause receptors to cleave off?

Postby Whelans2 » Jun 10 2016 1:53 pm

I heard the pure collagenases are not that effective overall?
Whelans2
technophile
technophile
 
Posts: 14
Joined: Jun 08 2016 1:50 pm

Re: Do Collagenases cause receptors to cleave off?

Postby sile88314 » Jun 12 2016 4:53 am

collagenases may degrade the collagen in extracellular matrix, so it is usually used in tissue/cell digestion. Do you want to isolate the membrane receptor or reduce the receptor degradation during the collagenase process? It is not simple to isolate a membrane protein as i know.
sile88314
technophile
technophile
 
Posts: 23
Joined: May 30 2016 1:21 am

Re: Do Collagenases cause receptors to cleave off?

Postby relaxin » Jun 12 2016 2:46 pm

Why do you want to use collagenases on T-cells? They are single cells. For isolation of cell surface receptors, you can use detergent.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7185
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: Do Collagenases cause receptors to cleave off?

Postby Whelans2 » Jun 15 2016 2:46 pm

I want to investigate if the low expression of the receptor is due to degradation caused during the collagenase process. I am just interested in T cells, I would be looking at tumours
Whelans2
technophile
technophile
 
Posts: 14
Joined: Jun 08 2016 1:50 pm

Re: Do Collagenases cause receptors to cleave off?

Postby relaxin » Jun 16 2016 9:38 am

Most collagenases used for cell isolation process are very crude and may contain other proteinases. So receptors on T-cells may be cleaved off by the proteinases. If you wash the isolated cells and let the T-cells recover for a day or two, their receptors may be regenerated. Comparing the receptors before and after recovery, you may tell low level expression of receptors is due to degradation during collagenase treatment or not.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7185
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: Do Collagenases cause receptors to cleave off?

Postby Whelans2 » Jun 24 2016 3:39 pm

Thanks for these responses. I did an experiment with different concentrations of collagenases at different time points. Im wondering what the best way to present it is. Was thinking expression of receptor on cell type against isotype control. Or just expression of receptors at different time points in one graph.
What are peoples suggestions?
Whelans2
technophile
technophile
 
Posts: 14
Joined: Jun 08 2016 1:50 pm

Re: Do Collagenases cause receptors to cleave off?

Postby relaxin » Jun 25 2016 7:50 am

Do you have a way to quantify the receptors? Since you have different concentrations and different time points, you can plot the receptor vs treatment time for each concentration of collagenase. The "0 time" control is "no treatment".
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7185
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: Do Collagenases cause receptors to cleave off?

Postby Whelans2 » Jul 12 2016 1:19 pm

Yes, I did this by flow but interestingly the isotype is quite high and im not sure why. Any ideas? Could collagenase do this?
Whelans2
technophile
technophile
 
Posts: 14
Joined: Jun 08 2016 1:50 pm

Re: Do Collagenases cause receptors to cleave off?

Postby relaxin » Jul 13 2016 10:15 am

It is difficult to tell, since the collagenase is not pure. Other contaminating enzyme(s) may be doing this.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
relaxin
PI of Posters
PI of Posters
 
Posts: 7185
Joined: Jan 11 2006 12:40 pm
Location: Mauna Kea

Re: Do Collagenases cause receptors to cleave off?

Postby Whelans2 » Jul 13 2016 4:55 pm

Thanks for the imput. Really appreciate it.

I found that the receptor is cleaved but ultimately we have no real way of predicting the extent of cleavage in tumor samples and on top of that, the expression level for the receptor of interest is so low. Perhaps the only way to be sure is to do a mechanical dissociation with the tumors. Although yield is low, we can be sure that the expression is preserved? Not sure if we have any other options? Also, perhaps we really need to focus on IHC
Whelans2
technophile
technophile
 
Posts: 14
Joined: Jun 08 2016 1:50 pm

Re: Do Collagenases cause receptors to cleave off?

Postby Whelans2 » Jul 14 2016 1:50 pm

I am thinking of testing the digestion cocktails that affected cleavage the least on the tumors we can get, from a range of indications (i.e. from low to high fibrotic content such as melanoma vs pancreatic). In addition to those cocktails, I would like to do a head to head with just mechanical on the same tumors (hopefully we would get enough tumor) and compare effects on i) expression and ii) cell yield and viability.

From my perspective, for the highly fibrotic tumors, we will definitely need some kind of digestion cocktail in there to free up the lymphocytes from the stroma. I don’t think mechanical will suffice. Thus, I’m hoping that in the context of actual tumor digestion, the effect of cocktails on cleavage will be somewhat reduced.

In addition to cocktails + mechanical, im thinking we could assess a cocktail that only consists of Collagenase IV + DNAse I (this is the cocktail I used in my postdoc to dissociate pancreatic tumors + soybean trypsin inhibitor). Would anyone have any additional comments or suggestions?
Whelans2
technophile
technophile
 
Posts: 14
Joined: Jun 08 2016 1:50 pm


Return to Immunology and Immunochemistry Methods

Who is online

Users browsing this forum: No registered users and 1 guest

cron