[Cytometry] whole blood **tech support response**

Roy Edward roy at biostatus.com
Mon Feb 17 05:55:51 EST 2014


Hello Barbara,
A couple of other alternatives might be ..

Lyse, no wash:
Björnsson, Sven, et al. "Total nucleated cell differential for blood and
bone marrow using a single tube in a five-color flow cytometer." Cytometry
Part B: Clinical Cytometry 74.2 (2008): 91-103.


This approach uses the hypotonic formic acid protocol for a lyse, no-wash
procedure.  The key step is the addition of the lysis solution, 5 second
vortex and then immediate addition of the neutralising buffer (author¹s
pers. comm.).  Nucleated cells are gated on the presence of the far-red
cell-permeant DNA probe, DRAQ5.  Additionally, back-gating on DNA content
for each phenotypically restricted population will then provide info on
cell cycle, aneuploidy, s-phase fraction, ..

No lyse, no wash:
Allan, Robert W., M. A. Ansari-Lari, and Sandra Jordan. "DRAQ5-based,
no-lyse, no-wash bone marrow aspirate evaluation by flow cytometry."
American journal of clinical pathology 129.5 (2008): 706-713.


This is an analysis of the merits and demerits of RBC lysis for flow
cytometry and compared to the classical microscopic method. To avoid any
of the risks associated with lytic reagent (most importantly lysis of late
erythroblasts and non-specific losses from wash steps), whole BM (and
equally peripheral blood, obviously) is diluted and stained with
antibodies and DRAQ5. Cells are gated on DNA content (also permitting
doublet exclusion).  Scatter properties are retained.

In either case, gating on a far-red DNA dye for nucleated cells is highly
sympathetic to the use of the new Brilliant Violet conjugates.

Kind regards,
Roy

Roy Edward
Email: roy at biostatus.com

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On 13/02/2014 14:10, "Harvey, Shane" <HarveyS at Princeton.Huntingdon.com>
wrote:

>Barbara,
>I have found that in the case of the lyse no wash protocol not working
>that going back the lysing with ACK Lysis buffer does the trick.
>
>Stain blood with your antibodies of choice for 15-30min
>Add 10:1 ACK/Blood volume of Lysis buffer
>Lyse for 8-12 min, then spin down for 5min at 400g
>Decant, Add your favorite buffer/1xPBS
>Spin 5min at 400g, decant.
>Add your Buffer/1xPBS Again, Spin for 5 min at 400g
>Decant, Fix, and you are good to go.
> 
>
>Shane Harvey
>Scientist
>
>Huntingdon Life Sciences Inc
>100 Mettlers Road 
>Somerset NJ 08873, USA
>
>HarveyS at Princeton.Huntingdon.com
><mailto:HarveyS at Princeton.Huntingdon.com>
> 
>
>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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