Cell migratory ability was detected by a modified wound healing assay. To assess the migratory abilities of PSCs, PSCs (5.0 × 105 cells/2.5 mL) were seeded into 6-well plates using DME/F12. To evaluate the migratory abilities of pancreatic cancer cells (PCCs) under coculture, Panc-1 (1.0 × 106 cells/2.75 mL) and PSCs (5.0 × 105 cells/2 mL) were seeded into the basolateral and apical sides of Millicell hanging cell culture inserts (pore size 0.4 μm) in 6-well plates, respectively, using a mixed culture medium (DMEM : DME/F12 = 1 : 1). After the cells grew to 90–100% confluency, a sterile pipette tip was used to produce a wound line between the cells in the plate. Cellular debris was removed by washing with PBS, and the cells were then allowed to migrate for 24 h. Images were captured at time 0 and 24 h post wounding under a Nikon Diaphot TMD inverted microscope. The relative distance traveled by the leading edge from 0 to 24 h was assessed using the Photoshop software (n = 3).
Diaphot tmd inverted microscope
Manufactured by Nikon
Sourced in Japan
The Diaphot TMD is an inverted microscope designed for a variety of laboratory applications. It features a stable, ergonomic design and advanced optical components to provide clear, high-quality images. The Diaphot TMD is suitable for various microscopy techniques, including brightfield, phase contrast, and fluorescence imaging.
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Lab products found in correlation
Embryomax ksom mouse embryo media, by Merck Group (1 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (1 mentions)
Folligon, by MSD animal health (1 mentions)
Megaclear kit, by Thermo Fisher Scientific (1 mentions)
Guide it recombinant cas9, by Takara Bio (1 mentions)
Guide it sgrna in vitro transcription kit, by Takara Bio (1 mentions)
Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Oil red o, by ScienCell (1 mentions)
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Glass coverslip, by Avantor (1 mentions)
Sf cell line 4d nucleofector x kit, by Lonza (1 mentions)
Gelatin, by Merck Group (1 mentions)
Trypsin edta solution, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
16 protocols using diaphot tmd inverted microscope
1
Quantifying Cell Migratory Ability
2
CRISPR-Cas9 Mediated Gene Editing in Mice
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C57CBAF1 female mice (7–8 weeks old) were superovulated by intraperitoneal injections of 5 IU of pregnant mare serum gonadotropin (PMSG, Folligon, MSD Animal Health) and an equivalent dose of human chorionic gonadotropin (hCG, Sigma) at a 48 hr interval. Superovulated female mice were mated with C57CBAF1 stud males and zygotes were recovered from oviducts.
Microinjections were performed with a micromanipulation system (Narishige MMO-202ND, MM-88) equipped on a Nikon Diaphot TMD inverted microscope. A mixture of 150 ng/µl of Cas9 mRNA and 50 ng/µl of sgRNA was delivered into the cytoplasm of zygotes (3–5 pl) using a filament needle (Bermejo-Álvarez et al., 2015 (link)).
Following microinjection, embryos were cultured in EmbryoMax KSOM Mouse Embryo Media (Millipore) at 37°C under 5% CO2 for 4 days until they reached the blastocyst stage. Six blastocysts were transferred to a pseudopregnant Swiss recipient 2.5 days post-coitum (dpc), resulting in the birth of two pups: one male carrying in-frame mutations and one founder female carrying an in-frame indel and a frameshift allele (KO allele). This F0 female was crossed with C57BL/6 males to obtain heterozygous mutants. Heterozygous F1 individuals harbouring the KO allele were intercrossed to produce WT, Hz or KO individuals used for the experiments.
Microinjections were performed with a micromanipulation system (Narishige MMO-202ND, MM-88) equipped on a Nikon Diaphot TMD inverted microscope. A mixture of 150 ng/µl of Cas9 mRNA and 50 ng/µl of sgRNA was delivered into the cytoplasm of zygotes (3–5 pl) using a filament needle (Bermejo-Álvarez et al., 2015 (link)).
Following microinjection, embryos were cultured in EmbryoMax KSOM Mouse Embryo Media (Millipore) at 37°C under 5% CO2 for 4 days until they reached the blastocyst stage. Six blastocysts were transferred to a pseudopregnant Swiss recipient 2.5 days post-coitum (dpc), resulting in the birth of two pups: one male carrying in-frame mutations and one founder female carrying an in-frame indel and a frameshift allele (KO allele). This F0 female was crossed with C57BL/6 males to obtain heterozygous mutants. Heterozygous F1 individuals harbouring the KO allele were intercrossed to produce WT, Hz or KO individuals used for the experiments.
3
RNA Microinjection for CRISPR-Cas9 Genome Editing
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Details for sgRNAs are provided on Table S3 . sgRNAs were synthesized and purified using Guide-it sgRNA In Vitro Transcription Kit® (Takara). Capped polyadenylated Cas9 mRNA was produced by in vitro transcription (mMESSAGE mMACHINE T7 ULTRA kit®, Life Technologies) using as template the plasmid pMJ920 (Addgene 42234) linearized with BbsI and treated with Antarctic phosphatase (NEB). mRNA was purified using MEGAClear kit (Life Technologies). A solution of 300 ng/µl of mRNA and 100 ng/µl of each sgRNA was used for RNA microinjection. For ribonucleoprotein injection, Guide-it Recombinant Cas9 (Takara) and sgRNA/s were mixed to a final concentration of 300 ng/µl and 60 ng/µl, respectively, and incubated at 37 °C for 5 min to achieve ribonucleoprotein assembly prior to microinjection. Previous experiments were conducted to determine that the concentrations used did not reduce developmental rates compared to sham (buffer) injections. Microinjection was performed under a Nikon Diaphot TMD inverted microscope delivering 3–5 pl into the ooplasm using a filament needle.
4
Dual Immunofluorescence Staining of TRPV1 and α1D-AR
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× 105/mL PC3 cells grown for 24 h at 37°C in poly-L-lysine coated slides, were permeabilized using 2% of paraformaldehyde with 0.5% of Triton X-100 in PBS and fixed by 4% of paraformaldehyde in PBS. After washes in PBS, cells were incubated with 3% of bovine serum albumin (BSA) and 0.1% of Tween-20 in PBS for 1 h at room temperature and then double stained with anti-TRPV1 (1:25) and anti-α1D-AR (1:25) Abs overnight at 4°C. Finally, samples were washed with 0.3% of Triton X-100 in PBS, incubated with Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated secondary Abs (1:100) for 1 h at 37°C and analysed with MRC600 confocal laser scanning microscope (BioRad, Hercules, CA) equipped with a Nikon (Diaphot-TMD) inverted microscope. Fluorochrome was excited with the 600 line of an argon-kripton laser. Serial optical sections were taken at 1-μm intervals through the cells. Images were processed using Jacs Paint Shop Pro (Jacs Sotfware Inc).
5
Quantifying Hyphal Fusion in Fungal Conidia
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Live-cell imaging was performed using an inverted microscope Diaphot-TMD inverted microscope (Nikon, Japan) to quantify CAT fusion activity for wild type and mutant strains as previously described [8] (link), [51] . In brief, 1 ml of fresh conidia at a density of 106 per ml were grown in Petri dishes (35×10 mm) at 34°C. Benomyl was added to distinguish CATs from germ tubes. This is because benomyl inhibits the formation of long germ tubes but not CAT formation or fusion [52] , [53] (link). After 4 hours of incubation, the cells were examined under the microscope for the presence of CATs and CAT fusion. For each sample, five random images were captured for later quantification of CAT fusion activity. The frequency of cell fusion observed in the mutant samples was compared to the wild type cell fusion frequency to obtain a relative CAT fusion activity for each of the mutants.
6
Wound Healing and Chemotaxis Assay
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A total of 3×105 cells from each cell line were plated in six-well plates. When the cells were confluent, an artificial wound was created using a 10 μL pipette tip 48 hours after transfection. Images were taken at two time points, 0 and 24 hours, using a Nikon Diaphot TMD inverted microscope (10×). In addition, cell migration was measured by chemotaxis. The lower compartment was filled with 30 μL epidermal growth factor (EGF), with different concentrations in each well (0, 1, 10, 100, and 1,000 ng/mL). In the upper compartment, 5×103 cells were added to each well. After 3 hours of incubation at 37°C, nonmigrating cells were removed and invading cells were stained with crystal violet. Three random fields were captured by microscopy at 20× magnification.
7
Lipid Staining and Quantification in Cells
8
Wound Healing Assay with BxPC-3 Cells
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BxPC-3 cells were seeded in 24-well plates (1.0 × 105 cells/500 μl). A sterile pipette tip was used to produce a wound line between cells after the cells grew to 90–100% confluence. Then, the cells were allowed to migrate for 24 h. Images were taken at time 0 and 24 h post-wounding using a Nikon Diaphot TMD inverted microscope (×10).
9
Fura-2 Calcium Imaging of Granule Cells
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Cultures were loaded with 10 μM Fura‐2‐ AM in incubation buffer for 30 min at 23°C. Cells were washed three times and mounted in a Warner Instruments field stimulation imaging chamber (RC‐21BRFS) on a Nikon Diaphot‐TMD inverted microscope. Images were acquired using alternate 340 nm and 380 nm excitation with 505 nm emission using a × 20 air objective and a Hamamatsu Orca‐ER CCD camera (Japan). Regions of interest were placed over granule cell neurites and their average pixel intensity values were calculated across each image sequence using the Time Series Analyser plugin from Image J (NIH). Data acquired at both 340 and 380 nm were expressed as a ratio and calibrated using the equation provided by (Grynkiewicz et al. 1985 ), where Rmin was obtained by addition of incubation medium supplemented with 1 mM EGTA and 10 μM ionomycin, Rmax by subsequent addition of saturating CaCl2 (5 mM) with the Kd for fura‐2 assumed to be 224 nM.
10
Quantifying Stella Maris Enzyme Activity
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After plants were treated with either 0.05%, 0.5% or 5% concentrations of Stella Maris® for 6 h, the plants were then washed with 50 mM sodium phosphate (pH 7), and 0.5 mL of GUS substrate solution (50 mM sodium phosphate, pH 7, 10 mM EDTA, 0.5 mM K4[Fe(CN)6], 0.5 mM K3[Fe(CN)6], 0.5 mM X-Gluc (5-bromo-4-chloro-3-indolyl-beta-d -glucuronic acid, cyclohexylammonium salt), and 0.1% v/v Triton X-100) was added to each well. The plants were then incubated at 37 °C for 4 h. Plant tissues were fixed with a 3:1 ethanol:acetic acid solution at 4 °C overnight and then placed in 95% ethanol. Plant tissues were cleared in lactic acid and examined using a Nikon DIAPHOT-TMD inverted microscope (Tokyo, Japan).
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