Flow cytometric discrimination of seven lineage markers by using two fluorochromes

<div><p>Flow cytometry is the primary immunological technique used to analyze multiple parameters on complex cell populations. We present a staining method that identifies major human mononuclear lymphoid and myeloid populations (CD4⁺ and CD8⁺ T cells, γδ T cells, B cells, NK cells and monocytes), using only two fluorochromes and a minimal number of cells. Our approach increases the number of markers recordable on most flow cytometers allowing for a deeper and more comprehensive immunophenotyping.</p></div

Two-fluorochrome immune-cell staining of cryo-preserved PBMC isolated from patients with multiple myeloma.

<p>PBMC isolated from patient with multiple myeloma involved in a clinical trial were collected and viable cryo-preserved at day 0, 14, 28, 60, 180 and 360 after stem cell transplant (SCT). (<b>a</b>) Frequency of the major lymphoid populations identified by the two-fluorochrome immune-cell staining. (<b>b</b>) Frequency over time of: naïve and memory CD8⁺ T cells; HLA-DR and CD57 memory CD8⁺ T cells; central memory (CM), effector memory (EM) and effector memory CD45RA (EMRA) CD8⁺ T cells. (<b>c</b>) Frequency over time of: naïve and memory CD4⁺ T cells; HLA-DR and CD57 memory CD4⁺ T cells; central memory (CM), effector memory (EM) and effector memory CD45RA⁺ (EMRA) CD4⁺ T cells; Th1, Th1/17, Th2, and Th17 CD4⁺ T cells (<b>d</b>) NK cell subsets.</p

Development and validation of the two-fluorochrome immune-cell staining strategy.

<p>PBMCs were isolated from a healthy donor and stained as described. (<b>a</b>) Lymphocytes were gated on the basis of their FSC-A and SSC-Area. To develop the final panel six steps were taken to incorporate a marker at the time as described in the text. Step 6 represents the complete array of lymphocyte populations that can be identified with the two-fluorochrome immune-cell staining. (<b>b</b>) Monocytes were gated on the basis of their FSC-A and SSC-Area and their flow cytometric profile with the complete two-fluorochrome immune-cell staining is shown. (<b>c</b>) PBMC were simultaneously stained with the two-fluorochrome immune-cell panel and with alternative antibody clones or markers conjugated with different fluorochromes directed against the same cell population. This data is representative of ≥ 10 experiments.</p

MOESM1 of Impaired B cell immunity in acute myeloid leukemia patients after chemotherapy

Additional file 1. Supplemental methods