Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
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