A nontoxic polypeptide oligomer with a fungicide potency under agricultural conditions which is equal or greater than that of their chemical counterparts

Article abstract of DOI:10.1371/journal.pone.0122095

There are literally hundreds of polypeptides described in the literature which exhibit fungicide activity. Tens of them have had attempted protection by patent applications but none, as far as we are aware, have found application under real agricultural c

Full text of DOI:10.1371/journal.pone.0122095

Application Information Bulletin: 13 colors on CytoFLEX
Advanced analysis of human T cell subsets on the
CytoFLEX flow cytometer using a 13 color tube based
on DuraClone dry reagent technology.
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1
Authors:
E-mail:
Nicole Weit , Michael Kapinsky , Andreas Böhmler
aboehmler@beckman.com
1
Affiliation:
Beckman Coulter GmbH, Krefeld, Germany
Beckman Coulter Immunotech S.A.S, Marseille, France
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Advanced analysis of human T cell subsets on the
CytoFLEX flow cytometer using a 13 color tube based
on DuraClone dry reagent technology.
PRINCIPAL OF THE TECHNIQUE
Summary
Multi-color flow cytometry is a powerful tool to analyze the highly heterogeneous human T cell compartment.
Using the 10 color DuraClone IM T Cell Subset dry reagent kit (CD45RA-FITC, CD197-PE, CD28-ECD,
CD279-PC5.5, CD27-PC7, CD4-APC, CD8-Alexa-Fluor* 700, CD3-APC-Alexa-Fluor* 750, CD57-Pacific Blue*,
CD45-Krome Orange) plus 3 additional liquid antibodies for the violet laser (Brilliant Violet 605TM anti-human
CD95, Brilliant Violet 650TM anti-human CD25, and Brilliant Violet 785TM anti-human CD127 antibodies) we
defined a 13-color tube which allows for the identification of major peripheral T cell subsets according to
classical and more recent characterization criteria, with a minimum of sample preparation effort.
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88 Excitation
638 Excitation
APC-
A750
405 Excitation
APC-
A700
Pacific Krome BV (3) BV
Blue* Orange 605
BV
785
FITC
PE
ECD PC5.5 PC7 APC
(
1)
650
(
2)
CCR7
CD197)
PD1
(CD279)
CD45RA
CD28
CD27 CD4
CD8
CD3
CD57
CD45
CD95 CD25 CD127
(
(
1) APC-Alexa Fluor* 700 (2) APC-Alexa Fluor* 750 (3) Brilliant Violet*
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INTRODUCTION
T lymphocytes (T cells) form an essential part of the adaptive immune system and are therefore of major
interest for the research community. Two recent examples are the work of the international ONE Study
(
www.onestudy.org) and BIO-DrIM (www.biodrim.eu) consortia, international groups of experts in the field
of immune monitoring, funded by the European Commission. Within these approaches, marker and dye
selections for flow cytometry have been designed and optimized by expert flow labs to monitor the human
immune response [1].
The close cooperation between these groups and Beckman Coulter resulted in the development of the
DuraClone IM brand of dry reagents for the analysis of human immune cells. Using the 10 color DuraClone
IM T Cell Subsets kit plus 3 additional liquid markers, a 13 color tube has been designed for the phenotypic
analysis of major T cell sub-populations in the human peripheral blood.
PROTOCOL
Standard Procedure:
After informed consent, 100 µL of human peripheral blood from a healthy donor was added to a DuraClone
IM T Cell Subset dry reagent tube (Beckman Coulter), followed by 5 µL each of Brilliant Violet 605TM
anti-human CD95, Brilliant Violet 650TM anti-human CD25, and Brilliant Violet 785TM anti-human CD127
antibodies (BioLegend). Cells were mixed for 8 seconds, incubated for 15 minutes at room temperature
(
RT) in the dark, and red blood cells were lysed by adding 2 mL of VersaLyse Lysing solution plus 50 µL of
IOTest 3 Fixative Solution (both from Beckman Coulter). Following incubation (20 min at RT), the suspension
was spun down (200 x g, 5 min), the supernatant discarded, and the pellet resuspended in 3 mL 1 x PBS.
After an additional centrifugation step (see above), the cell pellet was resuspended in 500 µL 1 x PBS for
subsequent analysis on a 13 color / 3 laser CytoFLEX flow cytometry system (Beckman Coulter).
CytoFLEX is the first commercial flow cytometer to utilize fiber array photo diodes (FAPD)s for fluorescence
channel detection. The FAPD provides low-noise detection with high quantum efficiency and minimum light
loss ensuring high signal to noise ratio and optical resolution especially with small particle measurements and
dim fluorescence detection.
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RESULTS
Analyses of T cell subsets based on the differential expression of surface molecules related to cell function, differentiation,
or activation have evolved [2]. As a result, T cell analysis requires a multitude of markers to capture the various populations
that have been described [3]. The 13 color tube presented here, allows for the identification of most T cell memory
subpopulations that can be characterized by surface marker expression patterns. It is suitable for all flow cytometers with a
5-3-5 (488 & 561 nm / 638 nm / 405 nm) optical layout and reduces sample preparation to basically only 4 pipetting steps.
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REFERENCES
1.
2.
3.
Streitz M. et al. Standardization of whole blood immune phenotype monitoring for clinical trials: panels
and methods from the ONE study. Transplantation Research 2013 2:17.
Appay V. et al. Phenotype and function of human T lymphocyte subsets: consensus and issues.
Cytometry A 2008 73A: 975.
Mahnke Y.D. et al. The who’s who of T-cell differentiation: Human memory T-cell subsets. Eur. J.
Immunol. 2013 43: 2797.
NOTES
The results demonstrated in this application sheet represent those generated on the Beckman Coulter
CytoFLEX Flow Cytometer with 488 nm / 638 nm / 405 nm laser configuration. 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
DuraClone IM T Cell Subsets Kit
VersaLyse Lysing Solution
Beckman Coulter
Beckman Coulter
Beckman Coulter
BioLegend
B53328
§
A09777 or IM3648
§
IOTest 3 Fixative Solution
A07800 or IM3515
Brilliant Violet 605 anti-human CD95
Brilliant Violet 650 anti-human CD25
Brilliant Violet 785 anti-human CD127
305628
302634
351330
BioLegend
BioLegend
§
Depending on geography.
For Research Use Only. Not for use in diagnostic procedures.
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CytoFLEX is a trademark of Xitogen Technologies (Suzhou), Inc., a Beckman Coulter company. CytoFLEX with
61 nm laser not yet available.
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*
Alexa Fluor and Pacific Blue are registered trademarks of Molecular Probes, Inc.; Brilliant Violet is a trademark of
Sirigen Group Ltd.
DuraClone, ECD and Krome Orange are trademarks of Beckman Coulter, Inc.
Beckman Coulter, IOTest, VersaLyse and the stylized logo are trademarks of Beckman Coulter, Inc and are registered
in the USPTO.
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FLOW-817APP03.15-A
© 2015 Beckman Coulter, Inc., All Rights Reserved