Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry

Abstract

Background: Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. Methods: Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. Results: All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying

Single and Coexpression of CXCR4 and CXCR5 Identifies CD4 T Helper Cells in Distinct Lymph Node Niches during Influenza Virus Infection

“…Single-cell suspensions of mediastinal lymph nodes (MedLN) from infected mice or inguinal lymph nodes from immunized mice were prepared as described previously (39). Cells were stained at 2.5 ϫ 10 7 cells/ml in "staining medium" (39), blocked with an anti-Fc receptor monoclonal antibody (MAb) (2.4.G2 at 5 g/ml) on ice for 15 min, and then washed and stained with antibodies against mouse CXCR5-biotin (BD Biosciences) and CXCR4-Alexa 647 (eBioscience) or CXCR4-allophycocyanin (BD Biosciences) at 37°C for 30 to 45 min.…”

Influenza Virus Infection Causes Global Respiratory Tract B Cell Response Modulation via Innate Immune Signals

“…For all experiments, propidium iodide (PI) was used at 1 g/ml in final medium to discriminate dead cells. Data acquisition was done using a FACSCalibur or FACSAria (BD Biosciences), the latter equipped with three lasers as described (34). FACS sorting and calcium-flux analysis was done using a MoFlo high-speed cell sorter (DakoCytomation), equipped with water-cooled lasers emitting in the blue (488 nm), red (647 nm), and violet (407 or 350 nm; the latter for calcium-flux analysis) and appropriate dichroics and bandpass filters for 11-color, 13-parameter analysis and sorting.…”

“…Loss of fluorescence intensity by live CD19 ϩ B cells was determined following staining with anti-CD19 (allophycocyanin) (eBioscience) and PI after 72-96 h culture. In vivo B cell proliferation was determined by measuring incorporation of BrdU in conjunction with multicolor flow cytometry as described elsewhere (34).…”

“…with 1 mg of BrdU and also received BrdU via drinking water for another 3 days. MLN and PLN were harvested on day 7 of infection and FACS analysis conducted for BrdU incorporation on CD19 ϩ B cells as described (34). Shown are results from three to five individual mice per group.…”

Severe interference between retinal angiography and automated four‐color flow cytometry analysis of blood mononuclear cells

“…These include, for example, the quantitation of expression of surface markers (10), such as CD38 on CD81 T lymphocytes in HIV infection (11), or CD20 on B cells in chronic lymphocytic leukemia (12), immunophenotyping of malignant cells in hematology (13), intracellular cytokine production (14), and enumeration of antigen-specific cytotoxic T cells using soluble major histocompatibility class I tetramers (15). More experimental investigations, such as P-glycoprotein-mediated transport of substances out of lymphocytes (16) or measurement of CD41 T cells proliferation by BrdU-FITC incorporation (17) are likely to be disturbed as well. Finally, in applications other than flow Figure 1.…”