title: Al adjuvants can be tracked in viable cells by lumogallion staining.
abstract: The mechanism behind the adjuvant effect of aluminum salts is poorly understood notwithstanding that aluminum salts have been used for decades in clinical vaccines. In an aqueous environment and at a nearly neutral pH, the aluminum salts form particulate aggregates, and one plausible explanation of the lack of information regarding the mechanisms could be the absence of an efficient method of tracking phagocytosed aluminum adjuvants and thereby the intracellular location of the adjuvant. In this paper, we want to report upon the use of lumogallion staining enabling the detection of phagocytosed aluminum adjuvants inside viable cells. Including micromolar concentrations of lumogallion in the culture medium resulted in a strong fluorescence signal from cells that had phagocytosed the aluminum adjuvant. The fluorescence appeared as spots in the cytoplasm and by confocal microscopy and co-staining with probes presenting fluorescence in the far-red region of the spectrum, aluminum adjuvants could to a certain extent be identified as localized in acidic vesicles, i.e., lysosomes. Staining and detection of intracellular aluminum adjuvants was achieved not only by diffusion of lumogallion into the cytoplasm, thereby highlighting the presence of the adjuvant, but also by pre-staining the aluminum adjuvant prior to incubation with cells. Pre-staining of aluminum adjuvants resulted in bright fluorescent particulate aggregates that remained fluorescent for weeks and with only a minor reduction of fluorescence upon extensive washing or incubation with cells. Both aluminum oxyhydroxide and aluminum hydroxyphosphate, two of the most commonly used aluminum adjuvants in clinical vaccines, could be pre-stained with lumogallion and were easily tracked intracellularly after incubation with phagocytosing cells. Staining of viable cells using lumogallion will be a useful method in investigations of the mechanisms behind aluminum adjuvants' differentiation of antigen-presenting cells into inflammatory cells. Information will be gained regarding the phagosomal pathways and the events inside the phagosomes, and thereby the ultimate fate of phagocytosed aluminum adjuvants could be resolved.
authors.affiliation: Department of Biomedical Science, Faculty of Health and Society, Malmö University, Malmö SE-205 06, Sweden.
authors.forename: Irene
authors.initials: I
authors.lastname: Mile
authors.affiliation: Lund Stem Cell Center, BMC B10, Lund University, Lund, Sweden; Division of Neurosurgery, Department of Clinical Sciences, Lund University, Lund, Sweden.
authors.forename: Andreas
authors.initials: A
authors.lastname: Svensson
authors.affiliation: Glioma Immunotherapy Group, Neurosurgery, Department of Clinical Sciences, Kampradbuilding barngatan 2b, Lund University, Lund SE-22185, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Darabi
authors.affiliation: Lennard-Jones Laboratories, The Birchall Centre, Keele University, Keele, Staffordshire ST5 5BG, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Mold
authors.affiliation: Glioma Immunotherapy Group, Neurosurgery, Department of Clinical Sciences, Kampradbuilding barngatan 2b, Lund University, Lund SE-22185, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Siesjö
authors.affiliation: Department of Biomedical Science, Faculty of Health and Society, Malmö University, Malmö SE-205 06, Sweden. Electronic address: hakan.eriksson@mah.se.
authors.forename: Håkan
authors.initials: H
authors.lastname: Eriksson
mesh_terms: D000276:Adjuvants, Immunologic / Q000493:pharmacokinetics*; D000536:Aluminum Hydroxide / Q000276:immunology / Q000493:pharmacokinetics*; D000537:Aluminum Oxide / Q000276:immunology / Q000493:pharmacokinetics*; D000818:Animals; D001557:Benzenesulfonates / Q000737:chemistry*; D045744:Cell Line, Tumor; D005419:Flavonoids / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D010587:Phagocytosis / Q000276:immunology; D010710:Phosphates / Q000276:immunology / Q000493:pharmacokinetics*; D013194:Staining and Labeling / Q000379:methods; D047309:Flavones
chemical_list: D000276:Adjuvants, Immunologic; D000536:Aluminum Hydroxide; D000537:Aluminum Oxide; D001557:Benzenesulfonates; D005419:Flavonoids; D005456:Fluorescent Dyes; D010710:Phosphates; C061331:4-chloro-3-(2,4-dihydroxyphenylazo)-2-hydroxybenzene-1-sulfonic acid; C069471:aluminum oxide hydroxide; C069096:aluminum hydroxyphosphate; C008548:morin; D047309:Flavones
keywords: Aluminum adjuvant; Flow cytometry; Lumogallion; Phagolysosome