[Cytometry] Help with macrophage detachment

Sean A Burke sab541 at nyu.edu
Fri Feb 6 11:46:52 EST 2009


I just came across this problem last week in my lab while trying to  
look at cell surface markers that are digested by trypsin. I found  
that 5mM EDTA in PBS w/o ca mg and supplemented with 0.2 %BSA works  
well. I chill the solution in ice for 30 minutes and apply it to the  
cells for 15 minutes while keeping the plate at 4 degrees, then I  
pipette gently to lift cells off the plate. I get great recovery and  
the cells look happy enough.  Hope this helps.


Sincerely,
Sean Burke
Department of Basic Sciences
New York University Dental Center
345 E 24th Street, S-921 New York, NY 10010
Phone: (212)-998-9276

On Feb 6, 2009, at 11:27 AM, Nebe-Von-Caron, G wrote:

> Has anyone tried to grow the macrophages on cygel? If I understand it
> right it is normally used to trap nonadherent cells but as it  
> dissolves
> when cooled it might release cells that grow on top of it as well.
>
>
> Gerhard Nebe-von-Caron
> Research Scientist and Biomedical Engineer
> SPDspark, www.swissprecisiondiagnostics.com
> SPD Development Company Limited
> Priory Business Park
> Stannard Way
> Bedford    MK44 3UP
>
> Company Registration No.  6032177
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>
>
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu
> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Paul W. Price
> Sent: 05 February 2009 18:45
> To: Parlane,Natalie; 'Wiener,Doris'; cytometry at lists.purdue.edu
> Subject: Re: [Cytometry] Help with macrophage detachment
>
> Microbiology Petri Dishes are very good, and so are the T flasks
> designed for suspension cultures (Sarstedt among others).  Having  
> still
> observed significant numbers of adherent cells using both, I found  
> that
> one or two rounds of ice-cold PBS (Ca++ and Mg++ free) containing  
> 0.2mM
> EDTA for 5 minutes improves recovery.
>
> Paul
>
> Paul W. Price, Ph.D.
> Director of R&D
> Abeome Corporation
> www.abeomecorp.com
> Tel. 706-542-7889
>
> -----Original Message-----
>> From: "Parlane, Natalie" <natalie.parlane at agresearch.co.nz>
>> Sent: Feb 3, 2009 7:30 PM
>> To: "'Wiener, Doris'" <dwiener at health.usf.edu>,
>> "cytometry at lists.purdue.edu" <cytometry at lists.purdue.edu>
>> Subject: Re: [Cytometry] Help with macrophage detachment
>>
>> Have you tried culturing in petri dishes (you know the common round  
>> (or
> square) ones used for microbiology culture)? They are sterile, the  
> MACs
> attach but then easily come off easily with a gentle tap. It was a  
> long
> time ago but you may need bring to room temp prior, too.
>> I believe the other tissue culture plates are coated to aid  
>> attachment.
>> Good luck
>> Natalie
>>
>>
>> Natalie Parlane
>> Research associate
>> Infectious diseases
>> Hopkirk Research Institute
>> AgResearch
>> New Zealand
>> T +64 6 351 8692
>> F +64 6 353 7853
>> E natalie.parlane at agresearch.co.nz
>>
>>
>> -----Original Message-----
>> From: cytometry-bounces at lists.purdue.edu
>> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Wiener,  
>> Doris
>> Sent: Saturday, 31 January 2009 4:15 a.m.
>> To: cytometry at lists.purdue.edu
>> Subject: [Cytometry] Help with macrophage detachment
>>
>> Thank you in advance for your help with this issue.
>>
>> I am trying to detach human monocyte derived macrophages from tissue
>> culture dishes. I have tried Sigma's Cell Detachment Media and
>> Genlantis' Detachin and have considerable cell death and up to 20% of
>> the cells still remain on the plate. I am hesitant to use trypsin/ 
>> EDTA
>> as I need to do surface staining of the cells for flow cytometry. I
>> have also tried mechanical removal (gentle scraping) but that also
>> caused a high percentage of dead cells. Thanks
>>
>> Doris Wiener
>> University of South Florida
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