The NPC cytosolic Ca2+ concentrations ([Ca2+]c) were measured by Fura-2 AM fluorescence (Invitrogen, Eugene, OR) on an Olympus IX70 inverted system microscope (Olympus USA, Center Valley, PA) equipped with a photometer and IPLab v3.71 software (Scientific Instrument Company, Campbell, CA), as previously described.6 (link) Briefly, NPCs were plated onto a 35mm laminin-coated culture dishes for 4 h, washed 3 times in Ca2+-free Dulbecco’s modified eagle medium (DMEM, Gibco, Grand Island, NY), loaded with 2.5 μm Fura-2 AM in the same buffer at 37 °C for 30 min, then washed and incubated in Ca2+-free DMEM for another 30 min at 37 °C. The cells were exposed to propofol (200 μM) in least essential medium (MEM, Gibco, Grand Island, NY) and fluorescence intensities were determined by recording at 340 and 380 nm excitation and at 510 nm emission, for up to 5 min, for each treatment. Data were presented as a ratio of 340/380 nm of fluorescence intensity normalized to baseline. After each imaging experiment, the trypan blue exclusion assay was routinely used to ensure that the cells for [Ca2+]c measurements were healthy and living.
Ix70 inverted system microscope
Manufactured by Olympus
Sourced in United States
The IX70 inverted system microscope is a versatile optical instrument designed for a range of laboratory applications. It features a modular design that allows for the integration of various accessories and components to suit specific research or imaging needs. The IX70 provides a stable and reliable platform for high-quality optical observations and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Bp460 490, by Olympus (1 mentions)
Hq 535 50m, by Chroma Technology (1 mentions)
Lc 20ad nano pump, by Shimadzu (1 mentions)
4 protocols using ix70 inverted system microscope
1
Measuring NPC Cytosolic Ca2+ Levels
2
Quantitative Analysis of Peritubular Capillaries and Glomerular Microvasculature
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To assess the number and size of PTCs, a CD31 costaining was used as previously described [18 (link), 19 (link)]. The PTC density was calculated by counting the total number of PTCs within the confines of each of 10 random 0.25 mm2 fields (each of these fields was delineated by a 1 cm2 ocular grid that was viewed at ×400 magnification), and the result is expressed as the mean per field. The diameter of these PTCs was also measured, and their mean was calculated in micrometers. To measure the PTC spaces, slides from each case were examined using an Olympus IX70 inverted system microscope (Olympus America, Melville, NY, USA) connected to a Hewlett Packard computer with Image-Pro Plus software (Media Cybernetics, Silver Springs, MD, USA) [20 (link)]. The intra-PTC area is expressed as the proportion of total PTC spaces over the entire cortex field. Intraglomerular capillaries were measured for their quantity (capillary counts per glomerulus) and diameter on cross-section of the glomeruli. To calculate the intracapillary inflammation cells, costaining of CD31 and inflammation markers (e.g., CD4, CD8, and CD68) were performed, and the results are expressed as the mean per PTC or glomerulus. Glomerular cross-sectional area was also determined using the Image-Pro Plus software.
3
Fluorescence Imaging of Coumarin and Amino Acids
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The mobile phase for coumarin analysis was water/acetonitrile (65:35, v/v) at a flow rate of 1 µL/min (LC-20AD nano pump, Shimadzu, Kyoto, Japan). In the case of amino acid analysis, we used water/acetonitrile/TFA (90:10:0.02, v/v/v) as the mobile phase. The flow path was switched using a high-pressure six-way valve (FCV-20H, Shimadzu). Fluorescence excitation was performed using a metal halide lamp. The filter cube was composed of an excitation filter (BP460–490, Olympus, Tokyo, Japan), a dichroic mirror (505DRLP, Omega Optical, Brattleboro, VT, USA), and an emission filter (HQ 535/50m, Chroma Technology, Rockingham, VT, USA). An IX70 inverted microscope system (Olympus, Tokyo, Japan) and an electron-multiplying charge-coupled device camera (iXon3, Andor Technologies, South Windsor, CT, USA) were used to observe the fluorescence images. Detection was performed near the outlet of the pillar array column.
4
Quantitative Analysis of EGFP Expression
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Cellular EGFP expression was quantitatively examined by FACS analysis and visualized using fluorescent microscopy. Cells were collected 48 h after Ad/VEGF-EGFP infection and approximately 10,000 cells were illuminated at 488 nm and fluorescence was detected in the FITC (525/20 nm) channel. Nonspecific fluorescence was detected using a 575/30 nm emission filter in the PI channel. EGFP fluorescence is expressed as the mean fluorescence signal in EGFP-positive cells in relative units [18 (link)] after subtraction of background fluorescence. An Olympus IX70 inverted microscope system (Olympus America, Melville, NY) was used for the screening of EGFP expression in cell monolayers.
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