Immunophenotyping: Instrument Calibration and Reagent Qualification for Immunophenotyping Analysis of Human Peripheral Blood Mononuclear Cell Cultures: Version 3

Excerpt

Immunophenotyping is the use of antigen expression for the identification of distinct immune cell subsets (and their activation statuses) [–4]. This technique can detect minute changes in cell populations and thus is used to characterize the cell makeup in many diseases as well as determine effects of treatments, such as nanoparticles [5]. It is important to develop a method that allows for immunological evaluation of nanoparticles because some nanoparticles are designed to modify the immune system while others cause immunotoxicity [4, 5]. Currently, the most common technique used to perform immunophenotyping is multicolor flow cytometry [2, 3].

NCL protocol ITA-37 covers two separate immunophenotyping panels (with 11-12 antibody-fluorophore conjugates):

1. Immunophenotyping Panel #1 (or Lymphocyte Panel):

   This panel includes antibody-fluorophore conjugates that allow for analysis of different lymphocyte populations including B cells and T cells (CD8+ T cells, CD4+ T cells, regulatory T (Treg) cells, naïve T cells, and γδ TCR T cells). This panel also determines cellular CD25 and CD154 expression which are markers of proliferation and co-stimulation/presentation, respectively.

2. Immunophenotyping Panel #2 (or Monocyte, Dendritic cell (DC), Natural Killer (NK)Cell Panel):

   This panel includes antibody-fluorophore conjugates that allow for analysis of different cell populations including CD14+ monocytes, DCs (plasmacytoid (p) and myeloid (m) DCs), and NK cells along with NK T cells. This panel also examines cellular CD69 and CD54 expression which are markers of early activation and adhesion, respectively.

When used in conjunction with other immunoassays, this protocol aids in establishing efficacy and safety profiles of engineered nanoparticles used for vaccine or drug delivery. This protocol has two parts, ITA-37.1 described in this document and intended for instrument calibration, and ITA-37.2, described in a separate document and intended for the analysis of nanoparticle-treated cells.