Fig 1.
Development and validation of the two-fluorochrome immune-cell staining strategy.
PBMCs were isolated from a healthy donor and stained as described. (a) 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) 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. (c) 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.
Fig 2.
Two-fluorochrome immune-cell staining of cryo-preserved PBMC isolated from patients with multiple myeloma.
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). (a) Frequency of the major lymphoid populations identified by the two-fluorochrome immune-cell staining. (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. (c) 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 (d) NK cell subsets.