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Basic questions

bascis FACS FSC SSC

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#1 madelingirly

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Posted 17 December 2014 - 11:56 PM

Dear Guys,

I urgently need your help

In our lab we have an old model of BD facscallibur, (http://www.rpciflow....ument Guide.PDF)

 

So please do you have any advice, manual or experience regarding how to really optimize the electronic setting before acquisition on this machine.

 

Another question, if I am using only one cell type, does gating is important.

 

After fixation and permeabilization of my cells, where should be my cells on FSC/SSC graph (Only one type of cells).

 

if I want to study membrane bound protein like N-cadherin, shall I do fixation and permeabilization.

 

if I want to study different proteins in the same cells ( and I have only green 2ry AB ) so I must divide the sample and stain separately with different 1ry AB then stain with the same 2ry AB and observe each sample alone, shall my FSC/SSC should be the same for these samples (the same cells the same experiment, but testing protein is different, so FL-1 should be different).

 

 

I am sorry if this is a basic questions, but I really would appreciate your help

Madelin

 

 



#2 Tabaluga

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Posted 19 December 2014 - 05:36 AM

Gating is important also with one cell type because you need to gate out debris and, if possible, doublets.

I worked with FACSCalibur, I don't really have any general advice but maybe if you have a particular problem...

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Il vivait. Il mourut quand il n'eut plus son ange;
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#3 Tabaluga

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Posted 19 December 2014 - 05:41 AM

Proteins where the epitope that is recognized by the AB is intracellular call for fixation/permeabilization. Surface proteins with extracellular epitope don't need it.

In your scenario, of course FSC and SSC should be the same in both samples - they are the same cells - if they got the same treatment regarding fixation etc. If FL-1 is for the green fluorescence then yes, it should be the only difference.

Hope it helps...

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 






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