title: Assessment of phagocytic activity of cultured macrophages using fluorescence microscopy and flow cytometry.
abstract: Phagocytosis is the process by which phagocytes, including macrophages, neutrophils and monocytes, engulf and kill invading pathogens, remove foreign particles, and clear cell debris. Phagocytes and their ability to phagocytose are an important part of the innate immune system and are critical for homeostasis of the host. Impairment in phagocytosis has been associated with numerous diseases and disorders. Different cytokines have been shown to affect the phagocytic process. Cytokines including TNFα, IL-1β, GM-CSF, and TGF-β1 were found to promote phagocytosis, whereas high mobility group box-1 (HMGB1) inhibited the phagocytic function of macrophages. Here, we describe two commonly used methods to assess the phagocytic function of cultured macrophages, which can easily be applied to other phagocytes. Each method is based on the extent of engulfment of FITC-labeled latex minibeads by macrophages under different conditions. Phagocytic activity can be assessed either by counting individual cells using a fluorescence microscope or measuring fluorescence intensity using a flow cytometer.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, 128 St. Albert Hall, 8000 Utopia Parkway, 11439, Queens, NY, USA.
authors.forename: Lokesh
authors.initials: L
authors.lastname: Sharma
authors.forename: Wenjun
authors.initials: W
authors.lastname: Wu
authors.forename: Sanjay L
authors.initials: SL
authors.lastname: Dholakiya
authors.forename: Samir
authors.initials: S
authors.lastname: Gorasiya
authors.forename: Jiao
authors.initials: J
authors.lastname: Wu
authors.forename: Ravikumar
authors.initials: R
authors.lastname: Sitapara
authors.forename: Vivek
authors.initials: V
authors.lastname: Patel
authors.forename: Mao
authors.initials: M
authors.lastname: Wang
authors.forename: Michelle
authors.initials: M
authors.lastname: Zur
authors.forename: Shloka
authors.initials: S
authors.lastname: Reddy
authors.forename: Nathan
authors.initials: N
authors.lastname: Siegelaub
authors.forename: Katrina
authors.initials: K
authors.lastname: Bamba
authors.forename: Frank A
authors.initials: FA
authors.lastname: Barile
authors.forename: Lin L
authors.initials: LL
authors.lastname: Mantell
mesh_terms: D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D008863:Microspheres; D010587:Phagocytosis / Q000187:drug effects*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D024243:HMGB1 Protein; C582808:HMGB1 protein, mouse; D053583:Interleukin-1beta; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016650:Fluorescein-5-isothiocyanate