Intravital microscopy was performed using LRP1ctrl and LRP1cKO mice as well as C57Bl/6 mice 2 h after intrascrotal injection of TNFα (500 ng per mouse) as previously described (Weckbach et al., 2014 (link)). For experiments with C57Bl/6 mice, anti-N-MK Ab (Cellmid) or the matching isotype control Ab (clone MOPC-21; 400101; BioLegend) were applied i.p. 12 h before the experiment. Briefly, after cannulation of the carotid artery, the cremaster muscle was surgically prepared as reported previously (Weckbach et al., 2014 (link)). Intravital microscopy was conducted using an upright microscope (Axiotech Vario, Zeiss; and DM6 FS, Leica) at 37°C and recorded using a digital camera (AxioCam HSm, Zeiss; and Zyla sCMOS, Andor Technology). Leukocyte counts were obtained from whole blood (ProCyte Dx; IDEXX Laboratories). Cells attached >30 s were defined as adherent. Diameter of postcapillary venules ranged from 20 to 40 µm. Centerline red blood cell velocities in microvessels were analyzed using fluorescent microspheres (0.5 µm diameter; Polysciences). Blood flow was calculated from the length of at least three microspheres measured in a snapshot image with a defined exposure time and converted offline to mean blood flow velocities. Wall shear rates were calculated as previously described (Weckbach et al., 2014 (link)).
Fluorescent microspheres
Manufactured by Polysciences
Sourced in United States
Fluorescent microspheres are small, spherical particles that emit light when exposed to a specific wavelength of light. They are typically made of polystyrene or other polymeric materials and are coated with fluorescent dyes. These microspheres are used in various applications, such as flow cytometry, cell tracking, and imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dm6 fs, by Leica (1 mentions)
Axiotech vario, by Zeiss (1 mentions)
Procyte dx, by IDEXX (1 mentions)
Clone mopc 21, by BioLegend (1 mentions)
Axiocam hsm, by Zeiss (1 mentions)
Epics altra 2 flow cytometer, by Beckman Coulter (1 mentions)
C 005, by Liposoma (1 mentions)
Polystyrene beads, by Merck Group (1 mentions)
5 protocols using fluorescent microspheres
1
Intravital Microscopy of Leukocyte Dynamics
2
Synechococcus Abundance Quantification by Flow Cytometry
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Abundances of Synechococcus were determined using 2-mL seawater aliquots on an Epics Altra II flow cytometer (Beckman Coulter, Brea, CA, United States) with a 306C-5 argon laser (Coherent, Santa Clara, CA, United States), according to the method of Jiao et al. (2002) (link). They were identified and distinguished from other autotrophs based on their positions in plots of side scatter versus red fluorescence and orange fluorescence versus red fluorescence. Event rates were set to 50–200 events/second (0.1–1 mL h-1) in order to enhance the particle capturing sensitivity. Fluorescent microspheres of 1 μm diameter (Polysciences Inc., Warrington, PA, United States) were added to all samples as an internal standard to calibrate flow rate and cell size. All samples were run in triplicate. The data were analyzed with EXPOTM32 MultiCOMP software (Beckman Coulter, United States).
3
Monocyte Adoptive Transfer Protocol
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Mice were challenged with 150 µl clodronate-containing liposomes (Liposoma, c-005) intravenously followed by 250 μl of fluorescent microspheres (Polysciences, 17154-10) intravenously injected 16–18 h later. GFP+ monocytes were subsequently purified and 1 million cells were injected into recipient mice.
4
Immunological Effects of WGP Exposure
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Mice were given a single 1 mg intraperitoneal dose of gently sonicated WGP, 3 μm polystyrene beads (Sigma-Aldrich), or 3 μm fluorescent microspheres (Polysciences) (all 1 mg in 200 μl of sterile PBS) or 200 μl of sterile PBS on day 0. For typically trained immunity studies, 7 days following the initial IP dose, mice were euthanized using CO2 and the pancreas along with other tissues of interest were removed and processed. For dose-titration studies, 0.5, 1, and 2 mg of WGP were delivered IP in 200 uL of sterile PBS. For time titration studies, mice were injected with 1 mg of WGP, and the pancreas was harvested after 24 h, 48 h, 3, 7, 10, 16, and 30 days later.
5
Chondrocyte Viability, Density, and Morphology Analysis
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Confocal projected axial views were analyzed using ImageJ/FIJI (National
Institutes of Health) and IMARIS software (Zurich, Switzerland) as described.12 (link)
Chondrocyte viability (% live cells) was calculated as: the number of
CMFDA-labeled cells/(number of CMFDA-labeled cells + number of PI-labeled cells)
in a given region of interest (ROI) volume. For chondrocyte density, the total
number of cells (CMFDA-labeled and PI-labeled) in the ROI volume were counted in
IMARIS, and results given as cells/μm3 (link)
. Chondrocyte volumes were obtained using the IMARIS “Surfaces” algorithm.
Volume calibration was performed using fluorescent microspheres (Polysciences,
Warrington, PA, USA). Chondrocyte morphology was considered “normal” if cells
were visualized as having a “smooth” surface and elliptical/rounded shape.
“Abnormal” chondrocytes exhibited at least one CMFDA-labeled cytoplasmic process
≥2 μm long. Abnormal cells were counted manually and divided by the total number
of live cells in the field of view with results presented as the % abnormal
cells in the whole cell population within the ROI.12 (link)
Institutes of Health) and IMARIS software (Zurich, Switzerland) as described.12 (link)
Chondrocyte viability (% live cells) was calculated as: the number of
CMFDA-labeled cells/(number of CMFDA-labeled cells + number of PI-labeled cells)
in a given region of interest (ROI) volume. For chondrocyte density, the total
number of cells (CMFDA-labeled and PI-labeled) in the ROI volume were counted in
IMARIS, and results given as cells/μm3 (link)
. Chondrocyte volumes were obtained using the IMARIS “Surfaces” algorithm.
Volume calibration was performed using fluorescent microspheres (Polysciences,
Warrington, PA, USA). Chondrocyte morphology was considered “normal” if cells
were visualized as having a “smooth” surface and elliptical/rounded shape.
“Abnormal” chondrocytes exhibited at least one CMFDA-labeled cytoplasmic process
≥2 μm long. Abnormal cells were counted manually and divided by the total number
of live cells in the field of view with results presented as the % abnormal
cells in the whole cell population within the ROI.12 (link)
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