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Does collagenase digestion require quenching?


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#1 Altair

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Posted 12 January 2015 - 11:46 AM

Hi all,

I'm planning to add an enzymatic digestion step with collagenase to my flow cytometry preparation protocol (to look at mouse lymph node dendritic cells). My planned procedure is the following:

1. excise LN
2. mince and incubate with collagenase D and DNAse I for 1h at 37C
3. filter through 70 um cell strainer
4. pellet and wash single-cell suspension with cold PBS
5. amine-reactive fixable live/dead stain, followed by normal antibody staining procedure (using PBS/0.1%BSA as my FACS staining buffer)

My question is, do I need to add anything to stop the collagenase digest at the conclusion of step 2? Or will the pelleting and PBS wash be sufficient to remove/stop the enzyme? (I know that for trypsin, one would add complete medium to quench the enzymatic activity, but am unfamiliar with collagenase).

Thanks for your help!


Edited by Altair, 12 January 2015 - 12:14 PM.


#2 Iceage

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Posted 12 January 2015 - 01:10 PM

After collagenase treatment, we usually wash cells with cold wash buffer (PBS/HBSS/DMEM, depending on the experiment), and keep the cells on ice after that. No quenching.



#3 Altair

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Posted 15 January 2015 - 12:19 PM

Thanks for the clarification!






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