[Cytometry] Opinions about planned experiment

Florian Uhle Florian.Uhle at chiru.med.uni-giessen.de
Wed Feb 17 13:57:55 EST 2010


Dear Flow cytometry community,

as I am a bloody beginner in the field of flow cytometry, I would like to hear your opinion on my planned experiment, which involves quantitative measurement of surface expression (I know, for a lot of flow operators this seems to be a no-go).

In a clinical study, I would like to measure expression level of a monocytic membrane receptor in the course of sepsis. To ensure comparability over time and between patients, I am trying to minimize all sources of variance:

-       	- instrument will be calibrated using CaliBRITE Beads before each measurement

-       	- antibody will be purchased in one large batch, directly conjugated from the providing company

-       	- isotype com will be purchased the same way

-       	- conjugate will be APC, buffer of antibody will be Tris/NaAz

-       	- upon arrival, antibody will be stored frozen in single experiment aliquots to avoid decrease intensity loss of fluorophore

-       	- staining protocol will be performed in whole blood; to avoid any Fc related binding, a preincubation with human IgG will be performed

-       	- readout should be geometrical mean fluorescence intensity

I would very much appreciate any comment on this, no matter if positive or negative. 

Best wishes,

Florian

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Florian Uhle
University Hospital Giessen
Department of Anesthesiology and Intensive Care Medicine
Rudolf-Buchheim-Straße 7
35392 Giessen

phone:   	+49-641- 99 444 77
fax:          	+49-641- 99 444 89
e-mail: 	florian.uhle at chiru.med.uni-giessen.de
URL:		http://www.uniklinikum-giessen.de/anaesthesie/
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