No-Lyse No-Wash Detection of CD45+ Leukocytes with the CytoFLEX Flow Cytometer

APPLICATION NOTE

Authors: Karen D. Carr and John Norman Affiliation: Beckman Coulter

IN THIS PAPER YOU WILL LEARN

To use of violet side scatter which works equally well for PBMC as side scatter

from 488 nm laser

The utility of a no-lyse, no-wash 8 color phenotypic assay

Introduction

Any researcher working with primary samples has a high regard for maintaining the integrity and natural state of their cell

population of interest. For phenotyping and some functional studies, maintaining a natural state of these primary samples

has to be considered in light of often rigorous lysing or separating techniques. If basic study of populations in peripheral

blood is to be performed, there is no need to lyse erythrocytes as staining can be done in whole blood. With whole blood

analysis, interrogation must be performed by a flow cytometer with electronics capable of handling concentrated samples

and a strong violet laser light source.

The CytoFLEX flow cytometer can sample from a minimum volume of 10 µL at rates up to 35,000 events per second making this instrument ideal for the analysis of concentrated samples such as unlysed whole blood. Additionally, the CytoFLEX has

an 80 mW violet laser that allows for powerful direct scatter detection of Violet side scatter off the hemoglobin carried

by erythrocytes1. Here, we test unlysed whole blood on the CytoFLEX flow cytometer with antibodies against a variety of

peripheral cell populations.

- 2 -

Methods

Whole Blood Staining

1. Fresh human blood sample was collected w EDTA.

2. 100 µL of patient sample was incubated with 5 µL of each of the following antibodies: CD45 Krome Orange, CD15 Pacific Blue, CD8 FITC, CD19 ECD, CD4 PC5.5, CD14 PC7, CD56 APC, and CD3 APC-AF750 for 15 minutes at room temperature in

the dark.

3. 20 µL of the stained whole blood was diluted into 180 µL of PBS and acquired on a CytoFLEX flow cytometer with a violet side scatter configuration. Threshold was set off the 500/40 CD45 Krome Orange channel.

Laser 405nm 488nm 638nm

Fluor(change if needed)

Krome Orange

Pacific Blue

V610 V660 V780 FITC PE ECD PC5.5 PC7 APCAPC

AF*700APC

AF*750

Filter (if different than standard configuration)

405/10

Marker CD45 CD15 Side scatter CD8 CD19 CD4 CD14 CD56 CD3

Clone J.33 80H5 B9.11 J3-119 13B8.2 RMO52 N901 UCHT-1

Figure 1. Unlysed Whole Blood is Well Characterized by Violet Light Scatter and CD45 (A). CD14+ and CD15+ cell populations

from leukocytes is depicted in (B). CD19 + and CD3+ lymphocytes are illustrated in (C). CD4+ and CD8 + T Cells are

characterized in D while CD3+CD56+ cells are easily discriminated in (E). Hierarchical classification is shown in (F).

* Alexa Fluor

A

ED

CB

F

© 2015 Beckman Coulter Life Sciences. All rights reserved. Beckman Coulter, the stylized logo, Kaluza are trademarks of Beckman Coulter, Inc. and registered in the USPTO. Krome Orange is registered trademark of Beckman Coulter, Inc. CytoFLEX and CytExpert are trademarks of Xitogen Technologies (Suzhou), Inc., a Beckman Coulter company.Alexa Fluor (AF) and Pacific Blue are registered trademarks of Molecular Probes, Inc.Brilliant Violet (BV) is a registered trademark of Sirigen.All other trademarks are the property of their respective owners.

FLOW-1308APP12.15-A.

For Research Use Only. Not for use in diagnostic procedures.

Results

Peripheral blood interrogated by flow cytometry in a native state with a No-Lyse No-Wash processing method allows for

clear discrimination of the subpopulations by scatter distribution. The relative abundance of the cellular subsets has not been

changed by centrifugation or “wash” steps.

References

1- Howard M. Shapiro. 1995. Practical flow cytometry. 3rd Edition, Wiley-Liss, Inc., New York.

Notes

The results demonstrated in this application sheet represent those generated on the Beckman Coulter CytoFLEX Flow

Cytometer. As differences exist in the performance between analyzers, the author cannot guarantee a similar appearance

with the use of other flow Cytometers.

Reagent Details

Reagent Supplier Order Details

CD15 Pacific Blue BCI A74775

CD45 Krome Orange BCI A96416

CD8 FITC BCI IM0451U

CD19 ECD BCI IM2708U

CD4 PC5.5 BCI B16491

CD14 PC7 BCI A22331

CD56 APC BCI IM2474U

CD3 APC-AF750 BCI A66329