Advanced analysis of human T cell subsets on the CytoFLEX flow cytometer using a 13 color tube-based DURAClone dry reagent
Content Type: Tech Note
Authors: Nicole Weit1, Michael Kapinsky2, Andreas Böhmler1
1 Beckman Coulter GmbH, Krefeld, Germany
2 Beckman Coulter Immunotech S.A.S, Marseille, France
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.
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 .
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.
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.
Analyses of T cell subsets based on the differential expression of surface molecules related to cell function, differentiation, or activation have evolved . As a result, T cell analysis requires a multitude of markers to capture the various populations that have been described . 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.
1. 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.
2. Appay V. et al. Phenotype and function of human T lymphocyte subsets: consensus and issues. Cytometry A 2008 73A: 975.
3. Mahnke Y.D. et al. The who’s who of T-cell differentiation: Human memory T-cell subsets. Eur. J. Immunol. 2013 43: 2797.
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.
|DuraClone IM T Cell Subsets Kit||Beckman Coulter||B53328|
|VersaLyse Lysing Solution||Beckman Coulter||A09777 or§ IM3648|
|IOTest 3 Fixative Solution||Beckman Coulter|| A07800 or§ IM3515
|Brilliant Violet 605 anti-human CD95||BioLegend||305628|
|Brilliant Violet 650 anti-human CD25||BioLegend||302634|
|Brilliant Violet 785 anti-human CD127||BioLegend|| 351330