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Improved Detection of Nicotinamide Adenine Dinucleotide Phosphate Oscillations Within Human Neutrophils

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Improved Detection of Nicotinamide Adenine Dinucleotide Phosphate Oscillations Within Human Neutrophils Technical Note Improved Detection of Nicotinamide Adenine Dinucleotide Phosphate Oscillations Within Human Neutrophils Andrea J. Clark,1 Roberto Romero,2,3,4 Howard R. Petty1,5* Abstract 1Department of Ophthalmology and Visual Kinetic studies of nicotinamide adenine dinucleotide phosphate autofluorescence have Sciences, The University of Michigan been conducted in adherent neutrophils using an improved microscopic photometry Medical School, Ann Arbor, MI system incorporating low noise excitation and detection systems. Dynamic autofluores- 2Perinatology Research Branch, Division of cence oscillations were found with periods ranging from 4 min to 10 s. The largest Intramural Research, Eunice Kennedy portion of the population of oscillating neutrophils (32%) had periods near 2 min. The Shriver National Institute of Child Health next largest group at 25% exhibited periods of 1 min or less. These oscillations could and Human Development (NICHD) of NIH, not be accounted for by instrument artifacts, cell shape changes away from the focal Bethesda, Maryland, and Detroit, Michigan plane, or other factors. They disappeared when detergent was added to oscillating cells. Higher-frequency oscillations disappeared as cells changed shape, indicating a correla- 3 Center of Molecular Medicine and tion between these two processes. This approach provides a reliable method to monitor Genetics, Wayne State University, this cellular property. ' 2010 International Society for Advancement of Cytometry Detroit, Michigan 4Hutzel Women’s Hospital at the Detroit Key terms Medical Center, Detroit, Michigan microfluorometry; autofluorescence; cell shape; metabolism 5Department of Microbiology and Immunology, The University of Michigan Medical School, Ann Arbor, MI MICROSCOPE-based photometry measurements, especially quantitative measure- Received 19 March 2010; Revision ments of fluorescence intensities, have been used for many years to study cell-associated Received 24 June 2010; Accepted 23 July labels or reaction product formation using fluorescence or absorption techniques (1). In 2010 addition to the application of exogenous substances to cells and tissues, endogenous cel- Grant sponsor: Division of Intramural lular materials can also be quantified using photometric tools, such as NADH autofluor- Research of the Eunice Kennedy Schriver escence. Microphotometry has also been extensively used to monitor dynamic changes National Institute of Child Health and within living cells based on the intensity of intracellular dyes using ratiometric methods, Human Development, NIH, DHHS; Grant such as in calcium and membrane potential measurements. Ratioing corrects for system- numbers: contract N01-HD-2-3342 and subcontract WSU04055. atic sources of error, including lamp fluctuations, dye loss, and sample thickness. For neutrophil metabolism studies, it is difficult to calculate the NADH/flavoprotein ratio *Correspondence to: Dr. Howard R. Petty, because the mitochondrial flavoprotein intensity is low and irrelevant flavoproteins, such Department of Ophthalmology and Visual Sciences, 1000 Wall Street, The as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (2), are present. University of Michigan Medical School, It is possible to ratio NADH fluorescence (470 nm) relative to a wavelength that is insen- Ann Arbor, MI 48105, USA sitive to metabolic activity, 415 nm (3), which controls for factors such as lamp fluctua- Email: [email protected] tions and cell shape/thickness changes. To measure the intensity of comparatively weak NAD(P)H autofluorescence, it is Published online 31 August 2010 in Wiley important to remove as much noise from the system as possible. Previously, our group Online Library (wileyonlinelibrary.com) developed a super-quiet microfluorometry system, which uses a single-photon counting DOI: 10.1002/cyto.a.20961 system in conjunction with a feedback-regulated super-quiet mercury–xenon lamp © 2010 International Society for driven by a highly stabilized power supply (4). Although this system works very well in Advancement of Cytometry most cases, including NAD(P)H detection in tumor cells, the ultraviolet intensity is not ideal for the detection of NAD(P)H autofluorescence in neutrophils. We have, therefore, used an ultraviolet light-emitting diode (UVILED) as a highly stable light source. This approach enabled us to conduct a quantitative survey of neutrophil NAD(P)H auto- fluorescence oscillations and to perform 450 nm/410 nm ratiometric analyses. Cytometry Part A 77A: 976À982, 2010 TECHNICAL NOTE MATERIALS AND METHODS mL; Pierce, Rockford, IL) in Hank’s balanced salt solution for 10 min at room temperature. Cells were washed and Materials then fixed a second time with 3.7% paraformaldehyde Phosphate buffered saline and low-intensity blue-fluores- (Sigma-Aldrich Chemical Co.). Dead cells did not display cent beads were obtained from Invitrogen Corp. (Carlsbad, motility or any normally observed movement, such as pseu- CA). Cover-glass bottom dishes were purchased from MatTek dopodia extension. Corporation (Ashland, MA). Unless otherwise noted, chemi- cals were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO). Microfluorometry System NAD(P)H autofluorescence was detected with micro- Isolation of Human Neutrophils fluorometry using a system similar to that previously Peripheral blood was collected from healthy male and described (4). As illustrated in Figure 1, light was delivered female donors in compliance with the guidelines of the Uni- from a continuous UVILED (365 nm) (Rapp OptoElectronic, versity of Michigan Institutional Review Board for Human Hamburg, Germany) to a Zeiss microscope via a liquid light Subject Research. Neutrophils were isolated from peripheral guide. Light was delivered to the sample using a 365WB50 ex- blood using Ficoll-Histopaque density gradient centrifugation citation filter and a 400-nm dichroic long-pass mirror (Sigma-Aldrich Chemical Co.). Samples were resuspended (Chroma Technology Corp., Rockingham, VT). Samples and then washed in phosphate buffered saline by centrifuga- were viewed using a 1003/numerical aperture 1.3 objective. A tion. After washing, cells were suspended in imaging buffer D-104 microscope photometer (Photon Technology Interna- (5) and placed in cover-glass bottom dishes. tional [PTI], Birmingham, NJ), containing a 450DF30 To provide a negative control, some purified neutro- emission filter, was connected to a refrigerated PMT housing phils were kept in vitro for 2 days before experiments to (Products for Research, Danvers, MA) containing an R1527P promote spontaneous apoptosis and cell death. In other photon-counting photomultiplier tube (Hamamatsu Corp., cases, neutrophils were fixed with a modified dithiobis(suc- Bridgewater, NJ). An adjustable diaphragm was used to cinimidyl propionate) procedure (6). In brief, cells were exclude other cells from the measurements and minimize the first fixed using dithiobis(succinimidyl propionate) (1 lg/ background intensity levels. All experiments were performed Figure 1. Microscope system diagram. A continuous ultraviolet light emitting diode (UVILED, 365 nm) source was attached to a Zeiss microscope via a liquid light guide. The light path is represented with a dashed line. The inset diagrams illustrate the configurations of fil- ters and mirrors. Light was delivered to the sample using a 365WB50 excitation filter and a 400-nm dichroic long-pass mirror (Chroma Technology Corp., Rockingham, VT). The signal was detected with a D-104 microscope photometer, containing a 440-nm dichroic reflector, a 450DF30 emission filter, and a 410-nm emission filter. Each emission pathway terminated at a refrigerated PMT housing containing a Hamamatsu R1527P photon-counting photomultiplier tube. For nonratiometric experiments, the 440-nm dichroic reflector was removed. An adjustable diaphragm was used to exclude other cells from the measurements. The attached computer used Felix software to collect and process data. All experiments were performed in a dark room within an aluminum enclosure with the microscope stage set to 378C. Cytometry Part A 77A: 976À982, 2010 977 TECHNICAL NOTE to the microscope photometer. A 440-nm dichroic mirror was inserted in the photometer to permit simultaneous emission measurements at two wavelengths. A 450DF30 optical filter (to detect NAD(P)H autofluorescence) and a 3RD millennium 400–420 nm discriminating filter (Omega Optical, Brattle- boro, VT), to detect changes in emission intensity independ- ent of cell metabolism, were used. Data Acquisition and Analysis Dishes containing neutrophils in imaging buffer were placed on a heated microscope stage and then were photo- graphed to document cell shape. Single-cell autofluorescence was recorded with a photomultiplier tube at four points per second in 20-min segments using FeliX software (PTI). In some cases, fluorescent beads, with excitation and emission properties similar to NAD(P)H were used. To minimize Brownian motion during observations, the beads were placed on slides, covered in low-melting-point agarose at 458C, then covered with a cover slip, and allowed to cool. After recording Figure 2. Data processing. Data were recorded at four points per experiments and controls, P values were calculated comparing second. Recordings are plotted in real time as intensity in counts per second versus time (min). Trace A shows raw data collected 10 consecutive raw data points at an oscillation peak with 10 from one recording. P values were calculated for adjacent peak
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