Three days after transfection of the CRISPR/Cas9 protein, cell pools treated with sgRNA targeting DPF3 or the nontarget control guides were used for the 3D-assay (3 wells per condition). In each well of a 48-well tissue culture plate, a mixture consisting of 93 μl Matrigel matrix (Standard Formulation, Corning) and 31 μl cell culture medium was dispensed and incubated for 30 min at 37 °C. Meanwhile, knockout and nontarget cell pools were trypsinized and counted. For each well, 10,000 cells were centrifuged and resuspended in 25 μl cell culture medium and 75 μl Matrigel matrix. Next, the suspension was added on top on the gel layer described above to avoid migration of cells to the bottom of the well. After incubation for 30 min at 37 °C, 1000 μl of cell culture medium was added. Medium was exchanged every 2 to 3 days. After 7 days, images were taken from each of the four quadrants per well using the Zeiss Primo Vert microscope and Zeiss Axiocam 105 camera (Zeiss Microscopy GmbH). Diameters of all captured spherical cysts were analyzed using ImageJ, and spherical cyst volumes were calculated according to 4/3πr3 (REF Nature communications).
Axiocam 105 camera
Manufactured by Zeiss
Sourced in Germany
The Axiocam 105 is a high-resolution digital camera designed for microscopy applications. It features a 5-megapixel CMOS sensor and offers fast image capture capabilities. The camera is compatible with a wide range of microscopes and can be used for various imaging tasks in research and industrial settings.
Automatically generated - may contain errors
Lab products found in correlation
Primovert microscope, by Zeiss (2 mentions)
Primovert, by Zeiss (2 mentions)
Matrigel matrix, by Corning (1 mentions)
Imager z1 apotome microscope, by Zeiss (1 mentions)
Zen 2, by Zeiss (1 mentions)
Axio microscope, by Zeiss (1 mentions)
Lsm 900 airyscan 2 microscope, by Zeiss (1 mentions)
Calcofluor white, by Merck Group (1 mentions)
Gelatin powder, by Merck Group (1 mentions)
Fd rapid golgistain kit, by FD NeuroTechnologies (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
Ms 222 solution, by Merck Group (1 mentions)
Zen lite 2012, by Zeiss (1 mentions)
10 protocols using axiocam 105 camera
1
3D Cyst Formation Assay for DPF3
2
Collagen-Embedded Cell Culture Assay
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plMDCK cells were resuspended in type I collagen, plated in 24-well plates and incubated at 37°C for 1 h as described previously. Then, medium supplemented with or without 10μM forskolin, 100μM ATP, 10μM ICA or 100 nM ACF was added and changed every 48 h. Images of four random visual fields per well were taken in a blinded manner at ×4 magnification using a Zeiss Primo Vert microscope and a Zeiss Axiocam 105 camera (Zeiss Microscopy GmbH, Jena, Germany) at day five.
3
Stability Analysis of Curcumin Nanoemulsion
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Polarization microscopy analysis was conducted using Carl Zeiss ApoTome Imager Z1 microscope (Zeiss, Göttingen, Germany), equipped with the AxioCam 105 camera and Zen Imaging software, in order to detect the presence of any larger droplets or curcumin crystals in the undiluted samples after two years of storage. A drop of the nanoemulsion formulation was placed on the microscope glass slide and captured under 400× magnifications.
4
Quantifying Cell Migration via Scratch Assay
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Cell monolayers were wounded with a sterile 200 μL pipette tip. Images of the cells were acquired at 0, 4, 8, and 12 h after scratching with an inverted microscope (Zeiss Primo Vert) equipped with the Axiocam 105 camera and ZEN 2.6 software (Carl Zeiss Inc.). The TScratch software was used to quantify open surface areas [23 (link)].
5
Imaging and Quantifying Fungal Cell Wall Glycans
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Pellets were imaged using a Zeiss Axiomicroscope equipped with an Axiocam 105 camera as described previously (33 (link)). β-(1-4) Glycans were stained with calcofluor white (Sigma) as described previously (9 (link), 11 (link)). Stack acquisition was done on a Zeiss LSM900 Airyscan 2 microscope. All fluorescent images were imaged with the same setting (laser intensity: 3.5%, pinhole: 47 μm, master gain: 750V, digital offset: −15, and digital gain: 1.0). For quantitatively comparing fluorescence, the measure region with the size of 15 μm by 15 μm squares at hyphal tips was used. Fluorescence was measured using ImageJ software (version 2.0.0/1.53c/Java 1.8.0_172/64-bit) (63 (link)).
6
Golgi Staining and Microscopic Imaging of Brain Samples
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Immediately following the completion of the MRI scan, animals were sacrificed by decapitation and brains were removed and a commercial Golgi staining kit (FD Rapid GolgiStain™ Kit; PK401, FD NeuroTechnologies Inc., Columbia, MD, United States) was used to stain the brain samples. In short, the removed brains were immersed in an impregnation solution. After 14 days of impregnation, the brains were frozen using isopentane and stored in a −80°C freezer.
The brains were then sectioned into 100-μm-thick coronal sections and stained in a multistep staining protocol on gelatin-coated slides (Objektträger 50 K, O. Kindler GmbH; Freiburg, Germany; gelatin powder, Sigma, St. Louis, MO, United States). The regions were imaged in bright-field mode under ×100 oil-immersion objective (EC Plan-Neofluar, NA 1.3) in z-stacks with an AxioImager Z2 microscope and AxioCam 105 camera (Carl Zeiss AG, Oberhocken, Germany). During the imaging process, the correct brain regions were identified, and then z-stacks were taken from each brain region (four z-stacks from both hemispheres), roughly from the same location within the area, keeping the order of the branch of the dendrite (second to third) constant. The imaging was performed blindly, so that the researcher did not know the treatment group of individual sections.
The brains were then sectioned into 100-μm-thick coronal sections and stained in a multistep staining protocol on gelatin-coated slides (Objektträger 50 K, O. Kindler GmbH; Freiburg, Germany; gelatin powder, Sigma, St. Louis, MO, United States). The regions were imaged in bright-field mode under ×100 oil-immersion objective (EC Plan-Neofluar, NA 1.3) in z-stacks with an AxioImager Z2 microscope and AxioCam 105 camera (Carl Zeiss AG, Oberhocken, Germany). During the imaging process, the correct brain regions were identified, and then z-stacks were taken from each brain region (four z-stacks from both hemispheres), roughly from the same location within the area, keeping the order of the branch of the dendrite (second to third) constant. The imaging was performed blindly, so that the researcher did not know the treatment group of individual sections.
7
Larval Development Quantitative and Qualitative Traits
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To follow the developmental changes occurring during larval development, we chose to identify both quantitative (eg, standard length, body depth, etc.) and qualitative (eg, appearance of fin rays, notochord flexion, pigmentation, etc.) (Table 1 ; Figure 4 B) traits. Pictures of at least three larvae (euthanized in a 200‐mg/L−1 MS222 solution; Sigma‐Aldrich) were taken daily from 1 dph to 21 dph, and at 30 dph under a stereomicroscope with brightfield‐transmitted and incident illumination (ZEISS V20 discovery‐Plan S objective 1.0 × equipped with an AxioCam 105 camera). Larvae were placed in a drop of filtered sea water on a Petri dish under the stereomicroscope. For each individual, a picture of the whole larva was taken at a 10× magnification to assess the eight selected quantitative traits. Measurements were performed using ImageJ software (V1.51k).79 All quantitative variables were analyzed using R,80 and we used SD to quantify the amount of variation within our sampling. Binary and multinomial logistic regression models were applied with the package “rms” on R to determine the best variable between age and SL explaining the qualitative criteria.81 To define the developmental stages, individuals were categorized according to the chronological appearance of the different qualitative criteria.
8
Wound Healing Assay in MCF-7 Cells
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pBABE control and pBABE miR-4521 over expressing MCF-7 cells at 90% confluency in a 6-well plate were synchronized by serum starvation for 48 h. Wound healing assay was performed by measuring the area of scratch healed at indicated time intervals. Images were captured using Zeiss Primovert microscope equipped with Axiocam 105 camera. Images were processed and analyzed using Zen v2.3 software. The rate of cell migration and migration index were estimated as per published protocols (Xu et al., 2012 (link)).
9
Invasion Assay of miR-4521 Overexpressing MCF-7 Cells
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0.5% low melting agarose solution mixed with 50 ng/mL EGF was spotted on 35 mm cell culture dishes. pBABE control and pBABE miR-4521 over expressing MCF-7 cells were seeded (8 × 104 cells) in the same dish containing agarose spots. After respective time points, the number of cells invading the agarose spots were counted and images were acquired using Zeiss Primovert microscope equipped with Axiocam 105 camera. Images were processed and analyzed using Zen 2.3 software.
10
Measuring Immune Encapsulation Response
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To evaluate immune defense, we measured encapsulation response as described in Lindstedt et al. (2011b ) after chemical defense measurements were taken. Larvae were first anaesthetized with CO2 (Lindstedt et al. 2018 ). A sterilized hypodermic needle was then used to puncture the skin on the dorsal part of the individual and a nylon implant (length 3 mm, diameter 0.11 mm) was inserted into the resulting hole. After 24 h, the implant was removed, dried, and photographed under a Zeiss DiscoveryV8 microscope equipped with an Axiocam 105 camera and ZEN lite 2012 software (Carl Zeiss Microscopy, LLC, Thornwood, NY). Every implant was photographed three times from three different angles (Rantala et al. 2000 ). Image analysis software ImageJ 1.47v (National Institutes of Health, USA) was then used to quantify the gray value of the implant, which was used as a measure of the encapsulation reaction. The gray value of the background was subtracted from the gray value of the implant to correct for potential variation in light source. The darker the implant, the stronger the immune response.
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