[Cytometry] whole blood

Simon Monard monard at wehi.EDU.AU
Wed Feb 19 17:55:10 EST 2014


Hi Barbara

I've always found the BD lysing solution works very well on peripheral blood. You are diluting the 10x solution with WATER right? for 100ul blood 900ul lysing solution does the job, you can use less if you want but not less than 500ul. 

Best

Simon



Simon Monard
Head of Flow Cytometry
Walter and Eliza Hall Institute
1G Royal Parade
Parkville Victoria 3052
Australia

Tel +61 3 9345 2541


----- Original Message -----
From: "Lucía Acosta" <acostalucia at gmail.com>
To: "barbara morandi" <morandibarb at yahoo.it>
Cc: "Purdue list" <cytometry at lists.purdue.edu>
Sent: Wednesday, 19 February, 2014 11:35:43 PM
Subject: Re: [Cytometry] whole blood

Bárbara,

Before you change your Lyse solution I recommend to you to check your protocol, for me the key for successfully Lyse red blood cells is the vortex, so after you stain and incubate your sample, and immediately you add your Lyse solution you should apply a gently vortex, then after 10 or 15 minutes (depends on your product) you will see that your sample becomes clear, then you should add PBS and wait 10 minutes before you acquire your sample.

For example I use 100 ul of blood after the incubation I add 500 ul of OptyLyse C then vortex and incubate  for 10 minutes, then I add 1 ml of PBS.

I used different flow cytometers and I can said that not all Lyse solutions works well in each instrument. So If you are using a BD Cytometer or a Cyan you can use the BD Lyse solution, Optylise B from Beckman or  Uti Lyse from Dako

If you are using a Beckman coulter Cytometer, you can use OptyLyse C

Also is important to check if your Lyse solution have a fixative solution, that is important if your are not acquire your samples immediately but is not recommended if you want to evaluate viability in that case is better that you use ammonium chloride (NH4Cl)

Regards 

Lucía Acosta  

> El 07/02/2014, a las 06:03, barbara morandi <morandibarb at yahoo.it> escribió:
> 
> Dear all,
> I would like to know whether someone can suggest how to perform immunofluorescence staining on whole blood (CD3, CD56, CD16, CD19, CD14). I've tried to lyse red cells with BD FACS lysis solution but it seems it doesn't work very well. Can anyone suggest a protocol to efficiently lyse red cells?
> Thanks a lot,
> Barbara
>  
> Dr. Barbara Morandi
> Immunology Unit
> Istituto Nazionale per la Ricerca sul Cancro
> CBA- L.go R.Benzi, 10
> 16132 - Genova, Italy
> Tel :39 (010) 5558-221
> Fax: 39 (010) 354282
> E-mail: morandibarb at yahoo.it
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