Cells were grown on 12 mm glass coverslips (Electron Microscopy Sciences, Hatfield, PA) in complete medium. Cells were fixed and permeabilized using 4% PFA and 0.1% Triton X-100 using standard methods. Cells were blocked for 30 minutes in 1% BSA. For nuclear size determination, cells were labeled in blocking buffer with 1:1000 mouse anti-Lamin B1 (AbCAM, Cambridge, MA) for 30 minutes at room temperature. Cells were stained for 30 minutes at room temperature in secondary antibody (goat anti-mouse Alexafluor488, Life Technologies). For cytoskeleton fluorescence, cells were stained with phalloidin-AlexaFluor647 (Life Technologies) and counterstained with DAPI according to the manufacturer’s instructions. ProLong Gold (Life Technologies) was used to mount the coverslips. Cells were imaged at room temperature using a Nikon Eclipse 90i upright fluorescent microscope and 10× or 20× objective (Nikon 10× PlanFluor DIC L/N1, 0.30 NA; Nikon 20× Plan Apo VC DIC N2, 0.75 NA). Images were captured using a Nikon Digital Sight DS-Qi1Mc camera at 1024×1024 or 1280×1024 pixel resolution. To quantify nuclear size, the images were thresholded using ImageJ. The “analyze particles” function was used to determine the nuclear area. At least 60 cells of each type were analyzed for nuclear size.
Digital sight ds qi1mc camera
Manufactured by Nikon
Sourced in United States
The Digital Sight DS-Qi1Mc is a CMOS digital camera designed for microscopy applications. The camera features a 1.45 megapixel resolution sensor and can capture images at a maximum resolution of 1392 x 1040 pixels. The camera is equipped with a C-mount interface for attachment to microscopes and has a global shutter mechanism for fast, precise image capture.
Automatically generated - may contain errors
Lab products found in correlation
Nis elements ar software, by Nikon (2 mentions)
A1r confocal microscope, by Nikon (2 mentions)
Te2000, by Nikon (2 mentions)
Axiovert 40c microscope, by Zeiss (2 mentions)
Phalloidin alexafluor647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
12 mm glass coverslips, by Electron Microscopy Sciences (1 mentions)
Mouse anti lamin b1, by Abcam (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Te300 epifluorescence microscope, by Nikon (1 mentions)
Csu 10 spinning disk confocal unit, by Hamamatsu Photonics (1 mentions)
Te2000 inverted microscope, by Nikon (1 mentions)
Nis elements ar, by Nikon (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
βiii tubulin, by Fortrea (1 mentions)
Eclipse ti s, by Nikon (1 mentions)
Alexa fluor 594 phalloidin, by BD (1 mentions)
Dylight 488 anti mouse igg, by Vector Laboratories (1 mentions)
Nis elements software, by Nikon (1 mentions)
Transwell insert, by Corning (1 mentions)
13 protocols using digital sight ds qi1mc camera
1
Immunofluorescence Imaging of Cell Nuclei
2
Live Cell Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Live cell microscopy was performed on a Carl Zeiss 200 m inverted microscope equipped with a Yokogawa CSU-10 spinning disk confocal unit, 100 × objective, 488 and 561 nm excitation lasers, and a Hamamatsu C9100-13 EMCCD detector. Any image panel featuring DAPI staining used fixed cells and was collected on a Nikon TE300 epifluorescence microscope equipped with a Nikon digital sight DS-Qi1MC camera using a 100 × objective and illuminated by LED. DAPI stained cells were prepared by inoculating 1 mL of YPD medium with cells from a yeast colony and incubating overnight. Cells were then sedimented by centrifugation at 3,000 g and resuspended in 100 μL of phosphate-buffered saline containing 4% formaldehyde. Fixation proceeded at room temperature for 15 minutes, after which cells were sedimented, washed with 1 mL tris-buffered saline (TBS), and again resuspended in 100 μL of a TBS solution containing 1% Triton X-100 and 200 ng/mL DAPI and incubated at room temperature for 30 minutes. Cells were again washed in 1 mL TBS before spotting on glass slides and imaging.
3
Time-lapse Imaging of Fungal Fate
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Time-lapse images for fungal fate analysis were captured on a TE2000 (Nikon) with Digital Sight DS-Qi1MC camera (Nikon), × 60 objective (DIC PLAN APO), using NIS elements AR software (Nikon). Transmitted light images were captured every 2 min and fluorescence images were captured every 10 min for 12 h. Two hundred and eighty-eight and 177 cells for AIg54 and AIg56, respectively, were analysed over four separate experiments. Time-lapse images for fungal fate analysis in the presence of apocynin were captured on a Ti (Nikon) with Neo (Andor), × 60 objective (DIC PLAN APO), using NIS elements AR software (Nikon). Transmitted light images were captured every 2 min and fluorescence images were captured every 10 min for 12 h. After completing the infection protocol, macrophages were treated with either 0.5 mM apocynin or 0.5% dimethyl sulphoxide (control). For strain AIg54 191 control and 129 apocynin-treated macrophages, and for strain AIg56 164 control and 148 apocynin-treated macrophages were analysed over three separate experiments. Three-dimensional imaging of mitochondrial tubularization was acquired with Nikon A1R confocal microscope (Nikon) in resonant scanning mode and with a piezo stage (MadCity Labs) for fast z-sectioning. C. gattii AIg54 was imaged in PBS containing 0.7 mM H2O2 to induce tubularization. Seventy-four z-sections were captured every 60 s for 40 min.
4
Time-lapse Imaging of Cryptococcus Phagocytosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After phagocytosis of cryptococci, chicken primary macrophages were washed at least five times in PBS before imaging. Time lapse images were captured on a TE2000 inverted microscope (Nikon) with Digital Sight DS-Qi1MC camera (Nikon), 20× objective (Ph1 PLAN APO), using NIS elements AR software (Nikon). Phase contrast images were captured every 2 minutes and fluorescence images were captured every hour for between 24 and 48 hours. The microscope was enclosed in a temperature controlled and humidified environmental chamber (Okolabs) with 5% CO2 at either 37 °C or 42 °C.
5
Quantifying Cellular Apoptosis and Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell cultures were fixed in 3% paraformaldehyde for 30 min at room temperature, then washed, and stored in phosphate-buffered saline (PBS). Apoptotic nuclei were detected by staining with Hoechst 33342, 1 μg/ml. Primary antibodies were rabbit anti-Ki67 (1:1000 Novcastra), β(III)tubulin (1:500, Covance), and rabbit-antiglial fibrillary acidic protein (GFAP; 1:500, DAKO).
Images were collected from random fields by a Nikon inverted fluorescent microscope (Nikon Eclipse Ti-S) equipped with a Nikon Digital Sight DS-Qi1MC camera. For quantitative analysis, the images were batch processed (to avoid bias) using the Volocity image analysis software (Demo-version, PerkinElmer) or ImageJ (http://imagej.net/ImageJ ).
Images were collected from random fields by a Nikon inverted fluorescent microscope (Nikon Eclipse Ti-S) equipped with a Nikon Digital Sight DS-Qi1MC camera. For quantitative analysis, the images were batch processed (to avoid bias) using the Volocity image analysis software (Demo-version, PerkinElmer) or ImageJ (
6
Monocyte Adhesion Assay with ICAM-1 Clustering
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ECs were grown to confluence on glass coverslips under normal growth conditions and treated with L-NIO (5 mM) ± [NTG (5 μM) or NTG-NL (5 μg/ml)] for 24 hr. Next, a monocyte adhesion assay was performed (as described earlier) and the U937 cell-EC cocultures fixed, permeabilized with 0.1% Triton X-100, blocked with 2% bovine serum albumin (BSA; Millipore, USA), and sequentially incubated with primary anti-ICAM-1 mouse antibody (Santa Cruz Biotechnology, USA) and secondary FITC-conjugated DyLight 488 anti-mouse IgG (Vector Labs, USA). To visualize actin microfilaments, U937 cell-EC cocultures were incubated with Alexa Fluor 594-Phalloidin (BD Biosciences, USA). Coverslips were mounted onto glass slides and fluorescence images (15 per condition) were taken using a Nikon Eclipse Ti microscope fitted with a Nikon Digital Sight DS-Qi1Mc camera. ICAM-1 clustering index was determined by measuring ICAM-1 fluorescence intensity (from n ≥ 10 images) at the U937-EC adhesion site and normalizing it to the average ‘background’ intensity measured from three neighboring EC cytoplasmic sites.
7
Immunofluorescence Localization of Toxoplasma Antigens
8
Fluorescent Labeling of Neuromuscular Junctions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tetramethylrhodamine (TMR) α-BgTX and fluorescein PNA were used essentially as first described by the laboratories of Cohen and Ko, respectively [34 (link),87 (link)]. TMR α-BgTX and Alexa-488 α-BgTX were purchased from Molecular Probes (Eugene, OR, USA) and used at concentrations and exposure times detailed in Results. Fluorescein-PNA was purchased from Vector Laboratories (Burlingame, CA, USA), and muscles were exposed to it at a concentration of 50 μg/mL for 20–30 min. f-PNA was originally used with R. pipiens NMJs, where it was shown not to adversely affect synaptic transmission [87 (link)]. We found that f-PNA also could serve in that capacity with X. laevis LP muscles (see Figure 13 B); in contrast, we found that f-PNA was not useful as a marker for NMJs in fish.
Frog and goldfish muscle preparations were imaged with a Nikon FN-1 (upright) microscope fitted with a Nikon Apo 40X/0.8 water-immersion objective and a Nikon Digital Sight DS-Qi1Mc camera using a 2-s exposure at a gain of 8. Zebrafish muscle preparations were imaged with a Nikon Ti (inverted) microscope fitted with Nikon Plan Fluor 40X/0.75 objective and a Retiga SRV-1394 camera (QImaging, Surrey, BC, Canada) with a 1-s exposure at a gain of 10. All images were acquired and processed with Nikon NIS-Elements software.
Frog and goldfish muscle preparations were imaged with a Nikon FN-1 (upright) microscope fitted with a Nikon Apo 40X/0.8 water-immersion objective and a Nikon Digital Sight DS-Qi1Mc camera using a 2-s exposure at a gain of 8. Zebrafish muscle preparations were imaged with a Nikon Ti (inverted) microscope fitted with Nikon Plan Fluor 40X/0.75 objective and a Retiga SRV-1394 camera (QImaging, Surrey, BC, Canada) with a 1-s exposure at a gain of 10. All images were acquired and processed with Nikon NIS-Elements software.
9
Transwell Invasion Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell invasion assays were performed using a transwell insert (Corning Incorporated, Corning, USA) with 4.4% Matrigel® (BD Biosciences, France) coated onto the transwell membrane (8 μm pore size, 6.5 mm diameter). The lower chambers of the transwell plates were filed with 700 μL DMEM/F12 containing 10% FBS. Cells were cultured in the higher chambers (200 000 cells/chambre for DAOY-MS and 1000 000 cells/chambre for D283-MS) onto 24-well plate with 500 μL serum free DMEM/F12. After 16 h for DAOY-MS and 48 h for D283-MS in incubator at 37°C, cells on the upper surface of filters were removed using cotton swabs and those on the lower surface were fixed 10 minutes with 4% paraformaldehyde and staining with 0.05% crystal violet of 30 minutes. Photomicrographs of whole culture surface were taken (Nikon AZ100, Digital Sight DS-Qi1Mc camera, Nikon, France). The coloration was solubilized 10 minutes by placing the inserts in 150 μL of 4% acetic acid and then transferred into 96 well plates, the absorbance was read at 540 nm with a spectrophotometer (Thermo Electron Corporation, Finland).
10
Fungal Fate Analysis via Time-Lapse Imaging
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!