Development of a 43 color panel for the characterization of conventional and unconventional T-cell subsets, B cells, NK cells, monocytes, dendritic cells, and innate lymphoid cells using spectral flow cytometry.

Although many flow cytometers can analyze 30-50 parameters, it is still challenging to develop a 40+ color panel for the phenotyping of immune cells using fluorochrome conjugated antibodies due to limitations in the availability of spectrally unique fluorochromes that can be excited by the commonly used laser lines (UV, Violet, Blue, Green/Yellow-green, and Red). Spectral flowcytometry is capable of differentiating fluorochromes with significant overlap in the emission spectra, enabling the use of spectrally similar fluorochrome pairs such as Brilliant Blue 515 and FITC in a single panel. We have developed a 43 color panel to characterize most of the immune subsets within the peripheral immune system, including conventional T cells, unconventional T cells such as invariant natural killer T cells (iNKT), Gamma delta (γδ) T-cell subsets (TCR Vδ2, TCR Vγ9) and mucosal-associated invariant T cells (MAIT), B-cell subsets, natural killer (NK) cells, plasmacytoid dendritic cells, dendritic cell subsets, hematopoietic progenitor cells, basophils, and innate lymphoid cell (ILC) subsets (CD117, CRTH2). The panel includes surface markers to analyze activation (CD38, HLA-DR, ICOS/CD278), differentiation (CD45RA, CD27, CD28, CD57), expression of cytokine and chemokine receptors (CD25, CD127, CCR10, CCR6, CCR4, CXCR3, CXCR5, CRTH2/CD294), and co-inhibitory molecules and exhaustion (PD-1, CD223/LAG-3, TIGIT), which enables a deep characterization of PBMCs from peripheral blood. Cells were analyzed on a 5-laser Cytek Aurora and data analysis was done using FlowJo. This panel can help to make a thorough interpretation of immune system, specifically when specimen quantity is low. The panel has not been completely optimized but would rather act as a guide toward the development of a workflow for optimized multicolor immunophenotyping panel.

Sahir F ，Mateo JM ，Steinhoff M ，Siveen KS ... -  《-》

OMIP-94: Twenty-four-color (thirty-marker) panel for deep immunophenotyping of immune cells in human peripheral blood.

This newly established 24-color (30-marker) panel focuses on the characterization of the main human immune cell subtypes and was optimized for the analysis of human whole blood using a full spectrum flow cytometer. The panel covers all main leukocyte populations: neutrophils, eosinophils and basophils, monocytes (with additional subsets), dendritic cells, innate lymphoid cells and lymphocytes. As for lymphocytes, this panel includes CD4+ T helper, Treg cells, and CD8+ cytotoxic T cells. Further T cells subsets are included with special focus on invariant T cells: γδ T cells (including δ2TCR variant), invariant NKT cells and MAIT (mucosal-associated invariant T cells) cells. Additionally, total B cells (including Bregs and plasmocytes), NK cells, and NKT cells are included. For the overall check of activation status of the analyzed immune cells we used HLA-DR, CD38, CD57, CD69, PD-1, and CD94. In addition, we used CD62L, CD45RA, CD27, and CD39 to describe the differentiation status of these cells. The panel was designed to maximize the information that can be obtained from surface markers in order to avoid the need for fixation and permeabilization steps. The presented multimarker panel offers the possibility to discover new immune cell subtypes which in patients and in cohort studies may lead to the identification of altered immune phenotypes and might give a link to immune system based or to certain other diseases. This panel was developed for a full spectrum flow cytometer equipped with a minimum of three lasers. We developed this panel using healthy human fresh blood, however it was also successfully used for staining of isolated human peripheral blood mononuclear cells (PBMC).

Pierzchalski A ，Zenclussen AC ，Herberth G 《-》

OMIP-084: 28-color full spectrum flow cytometry panel for the comprehensive analysis of human γδ T cells.

Using full spectrum flow cytometry, we designed a 28-color panel for the analysis of markers known to be associated with the γδ T cell immune response. This panel allows the classification of γδ T cell subsets via specific V gene usage (Vγ9, Vδ1, Vδ2, and Vδ3) of their T cell receptor (TCR) and according to their functional differentiation. Phenotypical surface receptors to distinguish different stages of cell maturation included CD45RA, CD27, CD28, CD127, CD57, and CD16; chemokine receptors CXCR6, CCR5, CCR6, and CX3CR1; NK-associated markers NKG2A, NKG2D, CD56, and CD161, checkpoint-inhibitor PD-1, and activating receptors CD38 and CD25. T cell lineage markers for the analysis of αβ T cells (CD4 and CD8) and MAIT cells (Vα7.2) were also included. This optimized multicolor panel allows a comprehensive immune-profiling of all main human γδ T cell subsets and is suitable for longitudinal or exploratory analysis of γδ T cell development and γδ T cell dynamics in clinical cohorts.

Barros-Martins J ，Bruni E ，Fichtner AS ，Cornberg M ，Prinz I ... -  《-》

OMIP-101: 27-color flow cytometry panel for immunophenotyping of major leukocyte populations in fixed whole blood.

This 27-color flow cytometry antibody panel allows broad immune-profiling of major leukocyte subsets in human whole blood (WB). It includes lineage markers to identify myeloid and lymphoid cell populations including granulocytes, monocytes, myeloid dendritic cells (mDCs), natural killer (NK) cells, NKT-like cells, B cells, conventional CD4 and CD8 T cells, γδ T cells, mucosa-associated invariant T (MAIT) cells and innate lymphoid cells (ILC). To further characterize each of these populations, markers defining stages of cell differentiation (CCR7, CD27, CD45RA, CD127, CD57), cytotoxic potential (perforin, granzyme B) and cell activation/proliferation (HLA-DR, CD38, Ki-67) were included. This panel was developed for quantifying absolute counts and phenotyping major leukocyte populations in cryopreserved, fixed WB collected from participants enrolled in large multi-site tuberculosis (TB) vaccine clinical trials. This antibody panel can be applied to profile major leukocyte subsets in other sample types such as fresh WB or peripheral blood mononuclear cells (PBMCs) with only minor additional optimization.

Imbratta C ，Reid TD ，Toefy A ，Scriba TJ ，Nemes E ... -  《-》

OMIP-099: 31-color spectral flow cytometry panel to investigate the steady-state phenotype of human T cells.

Zacharias ZR ，Houtman JCD 《-》