A general method for bead-enhanced quantitation by flow cytometry

Abstract

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry.

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

“…The numbers of MPs were detected by flow cytometry using a fluorescent bead counting assay. 24 By 24 hours, rotenone-induced apoptosis generated 6.7 Ϯ 0.1 ϫ 10 6 MPs, significantly more MPs than both untreated cells (0.2 Ϯ 0.1 ϫ 10 6 ; P ϭ 0.0001, n ϭ 3, Figure 2A) and 60°C HS-induced necrosis (2.7 Ϯ 0.1 ϫ 10 6 ; P ϭ 0.001, n ϭ 3, Figure 2B). By 48 hours, rotenoneinduced cells continued to produce significantly more MPs (13.3 Ϯ 0.2 ϫ 10 6 ) than untreated cells (0.2 Ϯ 0.1 ϫ 10 6 ; P ϭ 0.001, n ϭ 3, Figure 2A) or necrotic cells (2.1 Ϯ 0.2 ϫ 10 6 ; P ϭ 0.01, n ϭ 3, Figure 2B).…”

“…24 Briefly, 100 l of fluorescent beads (counted at 500/l with a hemocytometer) were added to 400 l of each supernatant and 300 l of dfPBS for a total volume of 800 l and the number of beads counted was stopped at 10,000. After subtracting the number of background fluorescent beads, the concentration of MPs was determined and the appropriate number of MPs was centrifuged (25,000 ϫ g, 1 hour at 4°C; in a SW40 Ti Swinging-Bucket Rotor in a Beckman L8-M, Class H, ultracentrifuge; Beckman Coulter).…”

CD4+Foxp3+ regulatory T cells mediate Toxoplasma gondii‐induced T‐cell suppression through an IL‐2‐related mechanism but independently of IL‐10

“…The following fluorochrome-conjugated mAbs were used: anti-CD3-FITC or -Cy5 (500A2); anti-CD4-TC, -PE or -APC (RM4-5); [63].…”

Chemokine Gene Expression during Fatal Murine Cerebral Malaria and Protection Due to CXCR3 Deficiency

“…Quantitation of BSLs was accomplished by spiking the samples with fluorescent microspheres (Invitrogen Life Technologies), at a concentration of 2.5 ϫ 10 4 /ml (43). Samples were analyzed using a FACSAria (BD Biosciences) and data analysis was performed using FlowJo.…”