Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages

Abstract

Blocking conditions that are optimal for the detection of surface antigens on resident peritoneal macrophages (PM+) by flow cytometry are not ideal for elicited or activated PM+. A blocking step of 10% goat serum can be used routinely to detect the F4/80 and Mac-1 antigens on resident PM+. In contrast, high concentrations (3350% each) of combined goat and mouse sera were required to reduce nonspecific binding and to improve the detection of the F4/80 antigen on PM+ elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE-2). However, even low concentrations of goat serum masked the expression of the Mac-2 antigen on TG and MVE-2 PM+. Thus, within a given elicited or activated PM+ population, different blocking conditions may be necessary to detect different surface antigens optimally. In addition to blocking, the use of isotypic controls that match the monoclonal antibody isotypes was found to be necessary for the optimal detection of antigen expression on TG and MVE-2 PM+. o

Inhibition of experimental autoimmune encephalomyelitis by a novel small molecular weight proinflammatory cytokine suppressing drug

“…Spinal cords were dissected from the vertebral canal. Mononuclear cells were isolated and prepared as previously described (Hendrzak et al, 1994; Pope et al, 1996). Data collection was performed on a LSR II (Becton Dickinson, San Jose, CA) flow cytometer using FACS Diva software (Becton Dickinson) and analysis was performed offline using FCS Express software.…”

CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

“…Spinal cords were dissected from the vertebral canal or removed by intrathecal hydrostatic pressure. Mononuclear cells were isolated and prepared as described previously [34,35]. Data collection was performed on a LSR II (Becton Dickinson, San Jose, CA, USA) flow cytometer in the Interdepartmental Immunobiology Center Flow Cytometry Facility (Northwestern University) using FACSDiva software (Becton Dickinson), and analysis was performed offline using FCS Express (De Novo Software, Los Angeles, CA, USA).…”

Phenotypic and functional characterization of pulmonary macrophages subpopulations after intratracheal injection of Paracoccidioides brasiliensis cell wall components

“…Since macrophage represent a special population of cells varying their shape, size and vacuolization throughout an inflammatory event (Hendrzak et al 1994) we established parameters (Silva et al 1997) to investigate PM using anti-CD16/32, anti-CD11b MoAb and Percoll discontinuous gradient. The differences among cell size, granularity and CD11b expression were better observed at 51 and 57% Percoll densities (Fig.…”

“…Positive staining was verified under fluorescence microscope (Nikkon ® ). Flow cytometry of macrophages was performed as described previously (Hendrzak et al 1994) using cell suspensions of 1 × 10 6 of BAL or PEM incubated at 4 • C for 30 min with 1 g of rat monoclonal anti CD11b (CD11b/CD18, BD-Pharmingen) anti CD23 (Mac-2, Boehringer, Mannheim) or anti Mac-3 antibodies (Accurate Chemical) anti-Ia d (BD-Pharmingen) diluted 1:100 in FACS-PBS containing 5% BSA, 8% FCS, 1% mouse control, 2.5% of normal goat or rat serum and 0.02% sodium azide. To develop the reaction, 10 l of anti-rat IgG, raised in goat and labelled with FITC (Dako) and diluted 1:2000 in the FACS-PBS was added and incubated at 4 • C for 30 min.…”