Live B. burgdorferi and complement are combined with serum. After incubation for 16 to 24 hr, B. burgdorferi are stained with acridine orange, which intercalates into double-stranded nucleic acids and produces green fluorescence (maximum emission 530 nm) when excited by incident laser light (488 nm). If the serum contains borreliacidal antibodies, acridine orange enters the spirochete and concentrates in blebbed surface areas characteristic of cell death. The spirochetes are accurately evaluated by flow cytometry and live (viable) and dead, blebbed (nonviable) B. burgdorferi are discriminated by the intensity of fluorescence.
Borrelia burgdorferi stock culture aliquots (see Support Protocol 3) in BSK medium
Barbour-Stoenner-Kelly (BSK) medium (see Support Protocols 1 and 2) Mueller-Hinton agar plates containing B. subtilis spores (see Support Protocol 4) Serum to be tested for anti-B. burgdorferi borreliacidal antibodies Normal antibiotic-free control serum from the same species as serum sample to be tested
Normal control serum containing 5 ^g/ml doxycycline (see recipe) Amberlite XAD-16 nonionic polymeric resin (Sigma) Phosphate buffered saline (PBS; appendix2a), pH 7.2, filter-sterilized Penicillinase (optional; Sigma)
Guinea pig complement (see Support Protocol 5), tested for activity (see Support Protocol 6)
Acridine orange working solution (see recipe)
50-ml screw-cap centrifuge tubes (e.g., Fisher) Filter paper
0.2-^.m microcentrifuge spin-filter tubes (1.5 ml; Costar) 56°C water bath
Petroff-Hausser counting chamber (Fisher) Dark-field microscope
12 x 75-mm polypropylene or polystyrene tubes as required for flow cytometer Flow cytometer with a 488-nm argon laser and 530/30 band-pass filters
Contributed by Steven M. Callister, Dean A. Jobe, and Ronald F. Schell
Current Protocols in Cytometry (2003) 11.5.1-11.5.12 Copyright © 2003 by John Wiley & Sons, Inc.
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