[Cytometry] Help with macrophage detachment

Cindy Zuleger clz at medicine.wisc.edu
Thu Feb 5 13:47:55 EST 2009


 When harvesting human monocyte-derived DC, I chill the plates for
20-30 min on a bed of crushed wet ice.  This seems to preserve viability
, surface expression of DC markers, and gets most (~90-95%) of the cells
off.  I also chill the wash buffer I am using to rinse the plates.  I
still need to do about 3 washes of the plates to get this yield.

Good luck!
>>> "Thure Adler" <thureroman at gmx.de> 02/05/09 8:00 AM >>> 
I once detached macrophages with the help of the product 'Accutase'
which is considered to be 'softer' than Trypsin/EDTA.
See this protocol also in: 
Heat shock protein 60 elicits abnormal response in macrophages of
diabetes- prone non- obese diabetic mice.
Adler T, Akiyama H, Herder C, Kolb H, Burkart V.
Biochem Biophys Res Commun. 2002 Jun 14;294(3):592- 6.

--------  Original- Nachricht --------
> Datum: Wed, 4 Feb 2009 13:30:34 +1300
> Von: "Parlane, Natalie" <natalie.parlane at agresearch.co.nz>
> An: "\'Wiener, Doris\'" <dwiener at health.usf.edu>,
"cytometry at lists.purdue.edu" <cytometry at lists.purdue.edu>
> Betreff: Re: [Cytometry] Help with macrophage detachment

> Have you tried culturing in petri dishes (you know the common round
> square) ones used for microbiology culture)? They are sterile, the
> 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
> 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,
> 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
> 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
> staining of the cells for flow cytometry. I have also tried
> removal (gentle scraping) but that also caused a high percentage of
dead cells.
> Thanks
> Doris Wiener
> University of South Florida
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Dr. med. vet. Thure Adler
Immunology Screen/GMC at the GSF
National Research Center for Environment and Health
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