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Fluorescence & Flow Cytometry Tutorial - (Jan/27/2009 )

Fluorescence & Flow Cytometry Tutorial

http://www.invitrogen.com/site/us/en/home/.../Tutorials.html

-Minnie Mouse-

Thank you, Minnie

-Nabi-

Minnie Mouse on Jan 27 2009, 05:41 PM said:




Thank you for the link. Its very informative

Bests

pooja

-pmaj-

if any body there can tell about how to calculate relative fluorescence units out of his to gram analysis in FACS. I want to know about mean, geometric mean and CV and their conversion into more understandable format of RFU and SD?

-gradbiotech-

Minnie Mouse on Jan 27 2009, 01:41 PM said:



great details, thanks...

-gradbiotech-

gradbiotech on Mar 8 2010, 09:46 AM said:

if any body there can tell about how to calculate relative fluorescence units out of his to gram analysis in FACS. I want to know about mean, geometric mean and CV and their conversion into more understandable format of RFU and SD?

I don't think converting the mean fluorescence as measured by the flow cytometer to RFU is possible, or rather, would make sense. As the name indicates, realtive fluorescence units are no really actual, universally defined units of measurement, but merely indicate the relative intensity of the measured fluorescence as compared to each other. The mean fluorescence measured by the flow cytometer, on the other hand, is the mean of the integrated areas of all light pulses measured by the detector, and is possibly instrument specific.

TL;DR I think that RFU and mean fluorescence are instrument- and experiment-specific values which are meaningless without controls to which they can be compared, and can therefore not be converted. Please correct me if I'm wrong.

-Aquifex-

gradbiotech on Mon Mar 8 07:46:20 2010 said:


if any body there can tell about how to calculate relative fluorescence units out of his to gram analysis in FACS.

First, you need calibration beads with known number of molecules of the fluorochrome of interest binded.

-Denis Baev-

I have to do Intracellular FACS mostly. Is it necessarry to Fc block receptors, I usually do all the staining in dark and in ice, to reduce the background.
So is it necessary, then why?

-Aindrila-

How do I get the absolute cell numbers for proper analysis?

-Aindrila-