< OMIPS Directory
OMIP-033

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

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

PURPOSE: LIMITED sample availability is a common problem for research with patient mate-rial, and this factor has hampered phenotypic studies. This work addresses the clearneed for a thorough immunophenotyping panel tailored for biological samples withfew cells. This panel is optimized for staining of both cerebrospinal fluid (CSF) lym-phocytes (containing 10–100,000 cells), as well as whole blood. The usually very lowCSF cell numbers make a one-step staining protocol necessary to minimize cell lossin washing steps. CSF cells must be stained immediately due to the CSF’s low proteincontent (1), which renders cells vulnerable. Ideal CSF samples are no older than 1 hand have no fewer than a total of 10,000 cells. This panel identifies human CD4 andCD8 memory subsets as well as T helper subsets, CD41 CD282 costimulation-independent T cells, B cell memory subsets, and plasma cells. Optimal whole bloodsamples should be no older than 5 h after sample acquisition (due to plasma cellloss). In these conditions, samples contain extremely few dead cells, and due to thenecessity 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 recom-mend inclusion of a live/dead marker, and also it needs to be noted that plasma cellsare sensitive to freeze/thawing.

CELL TYPE: CSF, Whole Blood-Fresh

MACHINE: BD LSR Fortessa

Marker Target Clone Fluorochrome Purpose
CD4 Human S3.5 PE-Texas Red T cell lineage
CD294 Human BM16 PE Th subset
CD194 Human L291H4 APC Th subset
CD3 Human Hit3a Alexa Fluor 700 T cell lineage
CD8 Human SK1 Brilliant Violet 510 T cell lineage
ccr7 Human G043H7 Brilliant Violet 421 naïve and memory subset
CD45RA Human HI100 Brilliant Violet 711 naïve and memory subset
CCR6 Human G034E3 Brilliant Violet 785 Th subset
CD19 Human HIB19 PerCP-Cy5.5 B cell
CD138 Human MI15 FITC Plasma cell
CD28 Human CD28.2 PE-Cy7 purative autoreactive cells
IgD Human IA6-2 Brilliant Violet 605 B cell
CD27 Human O323 APC-Cy7 naïve and memory subset