Fluorescence & Flow Cytometry Tutorial - (Jan/27/2009 )
Fluorescence & Flow Cytometry Tutorial
Thank you, Minnie
Minnie Mouse on Jan 27 2009, 05:41 PM said:
Thank you for the link. Its very informative
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?
Minnie Mouse on Jan 27 2009, 01:41 PM said:
great details, thanks...
gradbiotech on Mar 8 2010, 09:46 AM said:
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.
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.
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?
How do I get the absolute cell numbers for proper analysis?
I am trying to analyse pre-existing Flow Data for receptor expression, but a little confused about the controls. I believe they were all from tubes with equal amounts of cells/mL stained and a couple tubes unstained.
The controls are the confusing part...
1.) I have been given a set of Flow Cytometry data which has:
IC-PE (isotype control-pe), IC-FITC (isotype control FITC), ICAM-1and the unstained tubes.
From my understanding the unstained tubes work as my background/negative
I also been advised that my IC-FITC is also the background/negative
2.)But here is the confusing part, what is the IC-PE?
Is there a difference between IC-PE and IC-FITC? Are they both the background, treated as the negative?
3.)What does the ICAM-1 represent, would this be my positive control?
I also have other stained tubes which express positive for the receptors I am trying to find, so I do have a positive, but wondered why I have been given ICAM-1 as well?
4.)Also regards to MFI, which is best to calculate for MFI?
I have been using the median values of the IC-FITC, unstained and the tubes with the receptor staining to compare differences using an unpaired t-test, but have been advised this is incorrect.
Any clarification would be greatly appreciated please. Thank you.