Intracellular reactive oxygen species (ROS) were determined using 2′,7′-dichlorofluorescein diacetate (DCF-DA). RAW 264.7 cells were treated in the presence of 2 μg/mL lipopolysaccharide (LPS) together with samples at 37 °C for 1 h at a density of 2.0 × 105 cell/confocal dish. The increase of fluorescence signal was attributed to a self-oxidization process of DCF-DA, which transferred dichlorodihydrofluorescein (DCFH) to 2′,7′-dichlorofluorescein (DCF) with the presence of DCF. The fluorescent probe, DCF-DA, was used to monitor the intracellular generation of LPS-induced ROS. After 30 min of LPS treatment, DCF-DA 10 μM was added to the cells, which were subsequently incubated for 30 min at 37 °C. The intracellular ROS concentration was monitored using an inverted fluorescence microscope (Zeiss HBO 100, Thornwood, NY, USA).
Hbo 100
Manufactured by Zeiss
Sourced in Germany, United States
The HBO 100 is a high-pressure mercury short-arc lamp produced by Zeiss. It is designed to provide a high-intensity light source for various laboratory and research applications.
Automatically generated - may contain errors
Lab products found in correlation
Imager a2 microscope, by Zeiss (2 mentions)
Axiocam 503, by Zeiss (2 mentions)
Axiocam icc3, by Zeiss (2 mentions)
Axio imager m1, by Zeiss (2 mentions)
Axiocam 503 mono microscope, by Zeiss (2 mentions)
Axio imager a2 microscope, by Zeiss (2 mentions)
Axiovision rel 4, by Zeiss (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Efficient navigation zen , by Zeiss (1 mentions)
Alexa fluor 488 goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Axio scope a1 microscope, by Zeiss (1 mentions)
Infinite 200, by Tecan (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Plan neofluar, by Hamamatsu Photonics (1 mentions)
Axiovert, by Zeiss (1 mentions)
Zen software, by Zeiss (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Stemi sv11, by Zeiss (1 mentions)
19 protocols using hbo 100
1
Intracellular ROS Measurement by DCF-DA
2
Plasma Membrane Permeability Assay in Candida albicans
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Assessment of plasma membrane permeability was performed as described before [8 (link)], with modifications. Briefly, 3 mL of C. albicans cell suspensions (A600 = 0.1) in CP buffer (pH 6.0) were mixed with SDS (0–320 µg/mL), BAC (0–320 µg/mL) or Triton X-100 (0–320 µg/mL), incubated for 30 min at RT, washed three times with CP buffer, and stained for 5 min with PI to the final dye concentration of 6 x 10−6 M. Next, cell suspensions were washed twice with CP buffer and observed under a Zeiss Axio Imager A2 microscope equipped with a Zeiss Axiocam 503 mono microscope camera and a Zeiss HBO100 mercury lamp. The percentage of permeabilized cells was evaluated by counting PI positive cells out of one hundred cells in three independent repetitions for each experiment. Statistical significance analysis was performed using Student’s t-test (binomial, unpaired).
3
Visualization of Nanoparticle Distribution in Intestinal Mucosa
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The tissue distribution of nanoparticles in the gastrointestinal mucosa was visualized by fluorescence microscopy. For that purpose, 25 mg of Lumogen® F Red-labeled nanoparticles were orally administered to rats as described above. Two hours later, animals were sacrificed by cervical dislocation and the guts were removed. Ileum portions of 1 cm were collected, cleaned with PBS, stored in the tissue proceeding medium OCT™ and froZEN at −80 °C. Each portion was then cut into 5-µm sections on a cryostat and attached to glass slides. Finally, these samples were fixed with formaldehyde and incubated with DAPI (4′,6-diamidino-2-phenylindole) for 15 min before the cover assembly. The presence of both fluorescently loaded poly(anhydride) nanoparticles in the intestinal mucosa and the cell nuclei dyed with DAPI were visualized in a fluorescence microscope (Axioimager M1, Zeiss, Oberkochen, Germany) with a coupled camera (Axiocam ICc3, Zeiss, Oberkochen, Germany) and fluorescent source (HBO 100, Zeiss, Oberkochen, Germany). The images were captured with the software ZEN (Zeiss, Oberkochen, Germany). As control, a suspension of Lumogen® F Red 305 was administered.
4
Immunofluorescence Analysis of Endothelial Cells
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The cells were seeded onto 2% gelatin-coated coverslips in six-well plates. After reaching confluence, cells were fixed with 4% formaldehyde for 15 minutes and washed with PBS three times. The cells were then blocked in 5% bovine serum albumin (BSA) (Sigma-Aldrich) for 1 hour at room temperature and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 minutes. Cells were incubated with 5% BSA (negative control), antibodies against Von Willebrand factor (VWF) (Abcam6994, 1:400; Abcam, Cambridge, UK), or VE-cadherin (Abcam232880, 1:200) overnight at 4°C. Cells were washed with PBS three times and incubated with Alexa Fluor 488 Goat anti-Rabbit Secondary Antibody (Thermo Fisher Scientific) at room temperature for 30 minutes before mounting with ProLong Diamond Antifade Mountant and 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific). Five images were taken from randomly selected sections of cells grown on a coverslip using an immunofluorescence microscope (HBO 100; Carl Zeiss Meditec, Oberkochen, Germany) to calculate the purity of culture by dividing the VWF-positive cells of nuclei counts as previously described.28 (link)
5
Evaluating Fungal Cell Permeability
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The assessment of PM permeability was performed as described previously [53 (link)], with modifications. Briefly, 3 mL of Candida spp. cell suspensions (OD600 = 0.1) in PBS were mixed with ILs (concentrations equivalent to MIC90 against each fungi). Samples were incubated for 2 h at 37 °C, washed with PBS and stained with PI to the final dye concentration of 6 × 10−6 M for 5 min. Then, cell suspensions were washed with PBS and observed under the Zeiss Axio Imager A2 microscope equipped with the Zeiss Axiocam 503 mono microscope camera and Zeiss HBO100 mercury lamp. The percentage of permeabilized cells was evaluated by counting PI positive cells out of one hundred in three independent repetitions for each experiment.
6
Cartilage Mechanotransduction Via Elastography
7
Retinal Histomorphometry and Cellular Assays
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After the rats were euthanized, the eyeballs were immediately removed and fixed with FAS eyeball fixative. Subsequently, the eyeballs were embedded in paraffin, and cut into 5 μm thin slices along the vertical meridian of eyeballs. Then, the retinal tissues were stained with hematoxylin and eosin and photographed under a fluorescent microscope (HBO100, Zeiss). The retinal thickness measurement was extended from the optic disc to both ends, and each 50 μm was photographed. Three images were obtained in each direction, and the average value was calculated as the representative value of thickness. The thickness of inner nuclear layer (INL), outer nuclear layer (ONL), retinal pigment epithelium layer (RPE) and entire retinal layer was analyzed by the ImageJ software. One eyeball was taken from each of six rats in each group for analysis. Furthermore, images of TUNEL assay, ROS assay and immunohistochemical assay were obtained by photographing near the optic disc.
8
Microscopic Visualization of sfGFP Cells
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sfGFP expressed culture was centrifuged to prepare the sample. Collected cell was deposited on a glass slide and covered by cover glass. Each specimen was observed at 1000× magnification by ZEISS Axio Scope.A1 Microscope (ZEISS, Oberkochen, Baden-Württemberg, Germany) and Microscope illuminating system-HBO 100 (ZEISS).
9
DPPH Assay for Antioxidant Capacity
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The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to determine the anti-oxidant capacity of WCE. The DPPH radical scavenging activity is generally quantified in terms of inhibition percentage of the pre-formed free radical by anti-oxidants, and the SC50 (concentration required to obtain a 50% scavenging effect) is a typically employed parameter to express the anti-oxidant capacity. Ascorbic acid was used as an anti-oxidant standard to define the SC50 parameters. HaCat cells were incubated at a density of 2.5×104 cells in clear bottom black polystyrene 96-well plates (Costar, Corning, NY, USA) overnight. Cells were gently washed with Dulbecco’s phosphate-buffered saline (DPBS) 2–3 times and treated with WCE for 1 h. The fluorescent probe, dichlorofluorescein diacetate (DCF-DA), was used to monitor the intracellular generation of H2O2-induced ROS. After 30 min of H2O2 (1 mM) treatment, DCF-DA (total 1 μM) was added to the cells, which were subsequently incubated for 30 min at 37°C. The intracellular ROS concentration was monitored using a Zeiss HBO 100 (Thornwood, NY, USA) inverted fluorescence microscope and fluorescence microplate reader Infinite 200 (Tecan, Männedorf, Switzerland) at excitation and emission wavelengths of 485 and 535 nm.
10
Fluorescence Microscopy Protocol for Live-Cell Imaging
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Fluorescence microscopy was performed using an Axiovert 200 microscope equipped with a mercury lamp HBO100 (both from Zeiss, Jena, Germany) and appropriate filter sets (AHF Analysentechnik, Tübingen, Germany). Fluorescence emission was collected via a 40× Neofluar objective (Zeiss) and detected using a charge-coupled device (CCD) camera (Photometrics, Tucson, AZ, USA).
Live-cell experiments were performed in serum-free HBSS at 37 °C adjusted by an objective heating system (PeCon, Erbach, Germany) using a 100× α-Plan-Apochromat objective (NA = 1.46, Zeiss) on a modified Axiovert 200 microscope (Zeiss). For TIRF microscopy, the fluorescence of GFP or Cy5 was excited by a Kr+/Ar+ mixed gas laser (Innova, Coherent, Santa Clara, CA, USA) at 488 nm or 647 nm, respectively. Samples were illuminated in a TIRF configuration using a TIRF condenser (Till-Photonics, Gräfelfing, Germany) and a custom superflat beamsplitter (BS-zt488/647/780rpc). Emitted fluorescence was split into 2 channels using a beamsplitter (HC BS580), filtered using HC525/45 and ET700/75 bandpass filters, and imaged simultaneously on two CCD cameras (CoolSnap HQ, Photometrics, Tucson, AZ, USA). All filters were obtained from AHF Analysentechnik (Tübingen, Germany).
Live-cell experiments were performed in serum-free HBSS at 37 °C adjusted by an objective heating system (PeCon, Erbach, Germany) using a 100× α-Plan-Apochromat objective (NA = 1.46, Zeiss) on a modified Axiovert 200 microscope (Zeiss). For TIRF microscopy, the fluorescence of GFP or Cy5 was excited by a Kr+/Ar+ mixed gas laser (Innova, Coherent, Santa Clara, CA, USA) at 488 nm or 647 nm, respectively. Samples were illuminated in a TIRF configuration using a TIRF condenser (Till-Photonics, Gräfelfing, Germany) and a custom superflat beamsplitter (BS-zt488/647/780rpc). Emitted fluorescence was split into 2 channels using a beamsplitter (HC BS580), filtered using HC525/45 and ET700/75 bandpass filters, and imaged simultaneously on two CCD cameras (CoolSnap HQ, Photometrics, Tucson, AZ, USA). All filters were obtained from AHF Analysentechnik (Tübingen, Germany).
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