OMIPs Directory

OMIP-033

A comprehensive single step staining protocol for human T- and B-cell subsets

Cytometry PART A, Volume 89, Issue 7, 629-632 (2016)
Tess Brodie, Kristina Rothaeusler, Mireia Sospedra

Description: LIMITED sample availability is a common problem for research with patient material, and this factor has hampered phenotypic studies. This work addresses the clear need for a thorough immunophenotyping panel tailored for biological samples with few cells. This panel is optimized for staining of both cerebrospinal fluid (CSF) lymphocytes (containing 10–100,000 cells), as well as whole blood. The usually very low CSF cell numbers make a one-step staining protocol necessary to minimize cell loss in washing steps. CSF cells must be stained immediately due to the CSF’s low protein content, which renders cells vulnerable. Ideal CSF samples are no older than 1 hr and have no fewer than a total of 10,000 cells. This panel identifies human CD4 and CD8 memory subsets as well as T helper subsets, CD41/CD282 costimulation-independent T cells, B cell memory subsets, and plasma cells. Optimal whole blood samples should be no older than 5 h after sample acquisition (due to plasma cell loss). In these conditions, samples contain extremely few dead cells, and due to the necessity of a one-step staining, we did not include a live-dead cell discriminator. This panel can be successfully performed on frozen PBMCs, but authors then recommend inclusion of a live/dead marker, and also it needs to be noted that plasma cells are sensitive to freeze/thawing.

Species: Human

Tissue: Csf, Peripheral blood

Cell Subset: Plasma cell/ asc, B cell, T cell, T helper, T memory

Keyword: Cryopreserved pbmc

Machine: BD LSR Fortessa

Panel details

Marker Species Clone Fluorophore Purpose Co-expressions groups Antigen Density
CD294 Human BM16 PE Th subset T cell, T helper Med
CD194 Human L291H4 APC Th subset T cell, T helper Med
CD3 Human Hit3a Alexa Fluor 700 T cell lineage T cell, T helper, T memory High
CD45RA Human HI100 Brilliant Violet 711 naïve and memory subset T cell, T memory Med
CCR6 Human G034E3 Brilliant Violet 785 Th subset T cell, T helper Med
CD19 Human HIB19 PerCP-Cy5.5 B cell B cell Low
CD28 Human CD28.2 PE-Cy7 purative autoreactive cells T cell, T memory Med
IgD Human IA6-2 Brilliant Violet 605 B cell B cell High
CD27 Human O323 APC-Cy7 naïve and memory subset T cell, T memory, B cell Med
CD8a Human SK1 Brilliant Violet 510 T cell lineage T cell, T memory High
CD4 Human S3.5 PE-Texas Red T cell lineage T cell, T helper, T memory High
CCR7 Human G043H7 Brilliant Violet 421 naïve and memory subset T cell, T memory Low
CD138 Human MI15 FITC Plasma cell B cell Med