A subset of the rats (N = 9–11 per group) was used for measurements of retinal blood flow using microspheres, as we have described previously [13 (link)], and in accordance with the optimized procedure developed by Wang et al. [14 (link),15 (link)]. The animals were anesthetized with 100 mg/kg ketamine and 40 mg/kg pentobarbital, prior to the cannulation of the right femoral artery. An incision was made into the upper abdomen, which allowed the insertion of a 27-gauge needle through the diaphragm into the left ventricle. Fluorescent microspheres (2.5 × 106, 8 μm diameter; Bangs Laboratories, Fisher, IN, USA) were injected into the left ventricle while a reference blood sample was collected through the cannulated femoral artery. The rate of arterial withdrawal began 10 s before the injection and continued for a period of 30 s following the injection. The eyes were removed (and the rat euthanized with an overdose of pentobarbital), and the retinas of both eyes were prepared as flat mounts for counting the number of microspheres lodged in the retinal microvessels. The number of microspheres in the reference blood sample was counted with the use of a hemacytometer, and retinal blood flow rate was calculated as the femoral blood withdrawal rate multiplied by the ratio of microspheres found in the retinal tissue and divided by the number counted in the blood sample.
Fluorescent microspheres
Manufactured by Bangs Laboratories
Sourced in United States
Fluorescent microspheres are small, spherical particles that emit fluorescent light when exposed to specific wavelengths of light. These microspheres are designed for use in various applications, such as flow cytometry, cell tracking, and tracer studies.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Pd 1 apc, by BD (1 mentions)
Lag3 pe, by BD (1 mentions)
Cd8 pe cy5, by BD (1 mentions)
Annexin 5 fitc pi kit, by BioLegend (1 mentions)
Flash red, by Bangs Laboratories (1 mentions)
Cd3 apc cy7, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Dq gelatin, by Thermo Fisher Scientific (1 mentions)
Spectramax gemini xps, by Molecular Devices (1 mentions)
Prism 6, by GraphPad (1 mentions)
5 protocols using fluorescent microspheres
1
Measuring Retinal Blood Flow Using Microspheres
2
Comprehensive Immunophenotyping Protocol
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All flow cytometry antibodies were purchased from BD Pharmingen (San Diego, CA, USA), BioLegend (San Diego, CA, USA) and KeyGen Biotech (Nanjin, Jiangsu, China). The following antibodies were obtained from BD Pharmingen™: CD3-APC-Cy™7, CD4-FITC, CD8-PECy™5, PD-1-APC, and LAG3-PE. The following reagents were obtained from BioLegend: PerCP/Cy5.5-conjugated anti-IL-2, PE/Cy7-conjugated anti-IL-6, PerCP/Cy5.5-conjugated anti-TNF-α, and PE/Cy7-conjugated anti-IFN-γ antibodies, a FITC Annexin V/PI kit, and a KGA: FITC-BrdU kit. The following quantum MESF beads were purchased from Bangs Laboratories: Fluorescent Microspheres, Intensity Standard: Dragon Green, Flash Red, PE-MESF, and APC-MESF. The PMA/ionomycin mixture (250X) was purchased from MultiSciences (Lianke) Biotech (Hangzhou, Zhejiang, China). Enzyme-linked immuno sorbent assay (ELISA) kits for human interleukin-2 (IL-2), IL-6, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were purchased from Beijing 4A Biotech (Beijing, China). Brefeldin A was purchased from Qcbio Science & Technologies Co., Ltd. (Beijing, China).
3
Flow Cytometry Analysis of Tissue Factor Microparticles
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We measured TF‐positive microparticles with an SC MPL Quanta flow cytometer (Beckman Coulter) using published methods.31 The Quanta flow cytometer uses impedance to determine particle size, and fluorescence to detect TF. Fluorescent microspheres (0.78‐μm; Bangs Laboratories, Fishers, IN) functioned to calibrate particle size. Before quantification, MP samples were stained with Alexa Fluor 488–labeled monoclonal antibody (clone cH36) against human TF, or with Alexa‐labeled IgG antibody control (I4506, Sigma Aldrich). TF microparticles from human pancreas adenocarcinoma ascites metastasis‐1 pancreatic cancer cells served as a positive control.
4
Protease-Sensitive DQ-Gelatin Hydrogel Assay
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DQ-gelatin (Thermo Fisher Scientific) fluoresces in the presence of proteases. Stock solutions of DQ-gelatin were made according to the manufacturer’s instructions (1 mg/ml in diH2O). DQ-gelatin was co-encapsulated with Gel-HI hydrogels to a final concentration of 100 μg/ml. Fluorescent microspheres (Bangs Laboratories Inc.) or ECFCs were encapsulated in Gel-HI, and cluster formation was monitored via time-lapse microscopy (Zeiss AXIO Observer Z.1). DQ-gelatin fluorescence (488/515 nm excitation/emission) signal was continuously monitored in fluorescent microsphere experiments and quantified using ImageJ (public domain). Cell experiments required different hydrogel geometries; thus, fluorescence was measured at time 0 (T0) and time 24 hours (T24) using a SpectraMax Gemini XPS (Molecular Devices; Sunnyvale, CA) and graphed using GraphPad Prism 6 (GraphPad Software Inc.).
5
Micro-PIV Characterization of Laminar Flow
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To measure the velocity profile, micro-PIV techniques were used. Fluorescent
microspheres (polystyrene, diameter 1.0 μm, Bangs Laboratories,
Inc) were distributed in distilled water and sonicated to ensure uniform
particle dispersion. The flow was driven using a syringe pump through a channel
(height: ~200 μm, width 9mm) at a centerline velocity of
~0.5 mm s−1, corresponding to flow in the
laminar regime, Reynolds number (Re) of the order of 1, as shownSupplementary Fig. 6 . The fluid was
imaged under magnification starting below the lower SH surface and past the
upper non-SH surface. Upper surface can be determined by the presence of
in-focus, stationary, adsorbed particles on the glass coverslip, as can the
fluid/SH surface interface locations, however, due to the presence of air
pockets throughout, the lower surface was set at the fluid/air/surface interface
height. A set of 5 images spaced 30 ms apart were taken at each level,
processed to remove background noise and out-of-focus particles, and used to
generate an average velocity vector for the interrogation window (field of view)
per height plane. The pressure was obtained from a 12 cm long channel
with a height of 300um tested at four velocities.
microspheres (polystyrene, diameter 1.0 μm, Bangs Laboratories,
Inc) were distributed in distilled water and sonicated to ensure uniform
particle dispersion. The flow was driven using a syringe pump through a channel
(height: ~200 μm, width 9mm) at a centerline velocity of
~0.5 mm s−1, corresponding to flow in the
laminar regime, Reynolds number (Re) of the order of 1, as shown
imaged under magnification starting below the lower SH surface and past the
upper non-SH surface. Upper surface can be determined by the presence of
in-focus, stationary, adsorbed particles on the glass coverslip, as can the
fluid/SH surface interface locations, however, due to the presence of air
pockets throughout, the lower surface was set at the fluid/air/surface interface
height. A set of 5 images spaced 30 ms apart were taken at each level,
processed to remove background noise and out-of-focus particles, and used to
generate an average velocity vector for the interrogation window (field of view)
per height plane. The pressure was obtained from a 12 cm long channel
with a height of 300um tested at four velocities.
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