Whole blood method for simultaneous detection of surface and cytoplasmic antigens by flow cytometry

Abstract

Dual parameter analysis of surface antigens in flow cytometry has become a standard method for detection of cell subsets. However, few methods have been described for the extension of multiparameter analyses to include cytoplasmic or intracellular antigens. Here we describe a simple and reproducible method for simultaneous detection of surface and intracellular antigens by flow cytometry in lysed whole blood samples. This method employs the use of digitonin, a mild glycoside detergent, and formaldehyde for permeabilization and fixation. Red blood cells are lysed with 2% acetic acid. Preparation of samples in this manner resulted in altered light scatter characteristics relative to unpermeabilized samples; however, gating issues were overcome using a combination of scatter vs. fluorescence gating. Quantitation of CD3+/CD4+ and CD3+/CDS+ cells using this method was equivalent to counts obtained with the reference method using a commercially available lysis procedure and fluorescence vs. scatter gating. The effectiveness of the permeabilization process was assessed using a monoclonal antibody designated TIA-1, which is specific for a cytoplasmic antigen associated with cytotoxic granules predominantly found in CDS+ cells. The method effectively quantitated TIA-1 positive cells and demonstrated the specificity of the reagent for a subpopulation of CD8+ lymphocytes. Using this simplified procedure for simultaneous identification of surface and cytoplasmic antigens could help in studies of cell activation, proliferation, and other functional characteristics of the immune system.

Permeabilization and fixation conditions for intracellular flow cytometric detection of the T-cell receptor ? chain and other intracellular proteins in lymphocyte subpopulations

“…To further validate this permeabilization method, gated lymphocytes were evaluated for the expression of the cytolytic granule-associated protein detectable with the TIA-1 antibody. Consistent with the findings of Landay et al (14), this intracellular protein was found in few CD4-positive lymphocytes (Fig. 6A) and about half of CD8-positive lymphocytes (Fig.…”

“…Another in-house technique that works for detection of cytokeratin and proliferating cell nuclear antigen (PCNA), which permeabilizes and fixes cells via slow addition of ethanol to a final concentration of 60%, also was not successful for detection of the TCR chain. A published fixation/permeabilization procedure for detection of the 15-kD cytoplasmic granule-associated protein recognized by the TIA-1 antibody was not successful in detecting the TCR chain (14), but the technique described herein is able to detect the TIA-1 antigen as well as the TCR chain in T lymphocytes.…”

“…Although most of the chain is found in association with the TCR complex (31), some reports have suggested that the TCR chain may also interact with the transferrin (14) receptor (29) and/or the cytoskeleton (3). Detection of the chain in dual staining of T cells with a PE-labeled antibody to the CD3⑀ chain as described here allows the CD3⑀ chain to serve as a convenient internal standard for assessing TCR chain expression.…”

Formalin-fixed tumor cells effectively induce antitumor immunity both in prophylactic and therapeutic conditions

“…For flow cytometric analysis, most researchers have treated corresponding cells with formalin at concentrations less than 1-2% for 4 min at 37 • C prior to staining with antibodies in order to prevent in vitro degeneration [30,31].…”

Monitoring of monocyte functional state after extracorporeal circulation: A flow cytometry study

“…Detection of intracellular MPO was performed according to the Caltag (Burlingame, CA) protocol for cellular fixation and permeabilization (12)(13)(14) using the Fix & Perm cell permeabilization kit and appropriate volumes of anti-MPO (FITC-conjugated) monoclonal antibody (Caltag). An isotype control was carried out in parallel.…”