Forty-five thousand MPI-2 wild-type cells were seeded in a 96-well plate. On the following day, the virus was diluted in cold binding medium to reach 350 bound virus particles and added to the cells for 30 min. Five or 10 hours pi, cells were fixed with 3% PFA in PBS for 30 min at room temperature, washed twice with PBS, and dehydrated by subsequent incubation with 50, 70, and 100% ethanol for 2 min at room temperature. Dehydrated samples were stored at −20°C until staining. Samples were rehydrated by incubation with 70 and 50% ethanol and PBS for 2 min at room temperature. Rehydrated samples were FISH-stained against mouse Ccl2 mRNA using ViewRNA mRNA FISH assay (type 1 probe, Alexa Fluor 546, no. 6006661-01; probes were made against the sequence between mouse Ccl2 gene map positions 2 and 785; Thermo Fisher Scientific) according to the manufacturer’s instructions. Subsequently, cells were incubated in PBS containing DAPI and succinimidyl ester Alexa Fluor 647 (Thermo Fisher Scientific) for 10 min at room temperature. Cells were imaged in an ImageXpress Micro confocal microscope (Molecular Devices) (60-μm pinhole, 15 stacks, and 1.5-μm slice thickness) with a 40× objective. For quantification, cells were segmented according to the DAPI and succinimidyl ester signals, and segmented Ccl2 dots were then related to the cells using CellProfiler (70 (link)).
Imagexpress micro confocal microscope
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress Micro Confocal microscope is a high-performance imaging system designed for a wide range of applications in life science research. It is a fully automated, high-speed confocal microscope that captures high-resolution, multi-dimensional images of live or fixed cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Axio observer z1, by Zeiss (3 mentions)
Click it edu imaging kit protocol, by Thermo Fisher Scientific (2 mentions)
Facsdiva, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Black 96 well plate, by Greiner (2 mentions)
Metaxpress software, by Molecular Devices (2 mentions)
Fitc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Imagej, by Bio-Rad (1 mentions)
Goat anti rabbit fluorescein isothiocyanate fitc conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Methanol free paraformaldehyde, by Polysciences (1 mentions)
Rabbit anti ki67, by Merck Group (1 mentions)
Alexa fluor 568 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Black wall 96 well plate, by Corning (1 mentions)
Mitosox red, by Molecular Devices (1 mentions)
21 protocols using imagexpress micro confocal microscope
1
Quantification of Ccl2 mRNA Expression
2
Cell Cycle Profiling of Cancer Cells
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Investigation of the cell cycle profile of cancer cells was done using 5‐ethynyl‐2’‐deoxyuridine (EdU) labelling and the FUCCI system. Cancer cells were seeded (5 × 104 cells per well), treated with CTX for 3 days and allowed to recover in the absence of drugs either in MC or CC with fibroblasts. At selected time points, 10 µm of EdU was diluted in reduced serum media and added for 30 h to cultures. After the incubation time, cancer cells were fixed in 4% PFA for 20 min at RT and stained according to the Click‐iT EdU imaging kit protocol (Invitrogen, Waltham, MA, USA). Stained cancer cells were imaged and quantified using the ImageXpress Micro Confocal microscope (Molecular Devices, Sunnyvale, CA, USA). FUCCI system analysis was done using flow cytometry. Briefly, MCF7‐FUCCI cells were seeded and submitted to the same experimental setup described above. Cells were collected by trypsinization, and the channels RFP and GFP were acquired in BD FACS Canto II flow cytometer (BD Biosciences). Results were analysed using the BD FACS DIVA (BD Biosciences).
3
Cytotoxicity Screening of Epirubicin and Paclitaxel
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In a black 96‐well plate (Greiner Bio‐one, Kremsmünster, Austria), 1500–2000 cells per well were plated (day 0). At day 1, reduced serum medium with different concentrations of epirubicin (Biomol GmbH, Hamburg, Germany) and paclitaxel (Biomol GmbH) were added for 3 days. At defined time points, cells were imaged and analysed using the ImageXpress Micro Confocal microscope (Molecular Devices, Sunnyvale, CA, USA).
4
Microscopic Imaging of Cell Receptors
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Imaging of 24-well plates was executed with ImageXpress Micro Confocal microscope (Molecular Devices, San Jose, CA, United States), keeping the same exposure parameters constant (acquisition time and gain) between wells. The microscopic images were obtained by using MetaXpress software. The cell fluorescence was determined by the corrected total cell fluorescence (CTCF) method using ImageJ software (Schindelin et al., 2012 (link)). The localization and distribution of the HA-tagged receptors in 6-well or Ibidi plates were analyzed using a Zeiss LSM 710 (Oberkochen, Germany) confocal laser-scanning microscope.
5
GABA Receptor Distribution in Glutamatergic Synapses
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Immunofluorescence analysis was performed on synaptosomes as described previously [29 (link),30 (link)]. The synaptosomes were attached to the polylysine-coated coverslips for 40 min, fixed with 4% paraformaldehyde for 5 min, and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 1 h. Subsequently, the synaptosomes were incubated with primary antibody solutions containing anti-GABAB receptor antibody (1:100; Abcam, Cambridge, UK) and anti-vesicular glutamate transporter 1 (VGLUT1) antibody (1:100; Abcam, Cambridge, UK) overnight. Synaptosomes were then washed with PBS and incubated in a mixture of goat anti-mouse DyLight 549- and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. Immunoreactivity was visualized on a Image Xpress Micro confocal microscope (Molecular Devices, San Jose, CA, USA). The estimation of the percentage of glutamatergic terminals positive for GABAB receptor was counted three randomly selected areas (255 × 255 µm2) from each coverslip and averaged using ImageJ (Bio-Rad, Hercules, CA, USA).
6
Characterizing FPR Expression in HEK293T and U87 Cells
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Approximately, 2000 HEK293T cells or 3500 U87 cells were seeded in each well of a PDL-coated black optical 96-well μCLEAR-plate (Greiner Bio-One). Transient transfection of HEK293T cells was performed as described above. Twenty four hours after transfection, cells were fixated with 4% [v/v] methanol-free paraformaldehyde (Polyscience Inc) in PBS for 30 min at RT and afterward rinsed with PBS. After blocking with 5% [v/v] FCS in PBS for 30 min at RT, primary antibodies diluted in blocking solution were applied to the cells and incubated over night at 4 °C. Hereby, monoclonal antibodies for hFPR1 (R&D Systems, MAB3744, 1 μg/ml), hFPR2 (Santa Cruz Biotechnology, sc-57141, 0.2 μg/ml), and hFPR3 (R&D Systems, MAB3896, 1 μg/ml) were used. Cells were rinsed three times with PBS and subsequently treated with 2 μg/ml polyclonal alpaca anti-mouse antibody conjugated with Alexa Fluor 568 (Invitrogen) and 2 μM Hoechst 33342 (Thermo Fisher) for 60 min at RT. Image acquisition was performed with a Molecular Devices ImageXpress Micro confocal microscope and analyzed using MetaXpress software (Molecular Devices).
7
Osteoblast Proliferation Assay by Ki67 Staining
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The Ki67 staining was performed as previously described.(47 ) Briefly, the primary osteoblasts isolated from long bones were plated on 0.32 cm2 of each well in a 96‐well plate (6.4 × 103 cells/well). After 48 hours of seeding, the culture medium was replaced with osteogenic induction medium. At day 17, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X‐100 (cat. X100; Sigma) for 10 minutes, blocked with 30 μL of IHC select blocking buffer (cat. 20773; Merck) for 30 minutes, and stained with rabbit anti‐Ki67 (cat. Ab15580; Abcam, Cambridge, UK) antibody (1:100 in blocking buffer) (1 hour; RT). The cells were washed thrice in PBS and 30 μL of Alexa‐488 anti‐rabbit secondary antibody (1:400 in blocking buffer) (cat. A21206; Thermo Fisher Scientific) was added (45 minutes; RT). The cells were washed again thrice with PBS followed by DAPI staining (1 μg/mL) (cat. 62248; Thermo Fisher Scientific) (5 minutes; RT). The samples were imaged using ImageXpressMicro confocal microscope (Molecular Devices, San Jose, CA, USA)(47 ) and were analyzed using cell profiler software.(55 )
8
Histamine-Induced Cytoskeleton Remodeling
9
Immunofluorescence Assay for p8 Protein in Colorectal Cancer Cells
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Colorectal cancer (DLD-1) cells were seeded onto coverslips placed in 6-well plates. After 24 h, p8 protein (0–40 μM) was added to each well for a further 72 h. Cells were fixed for 15 min at RT in 3% paraformaldehyde (PFA) and then washed three times in PBS. Cells were permeabilized by incubation for 2 min in 0.2% Triton X-100/PBS and then washed. To reduce background signals, cells were blocked for 30 min with 4% bovine serum albumin (BSA) in PBS. Next, cells were incubated overnight at 4 °C with a rabbit polyclonal anti-p8 antibody (Young In Frontier Co., Ltd) or for 2 h at 4 °C with a mouse monoclonal anti-EpCAM antibody (Cell Signaling Technology). Protein localization was visualized using FITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.; West Grove, PA, USA) and Alexa Fluor 568-conjugated donkey anti-mouse IgG (Invitrogen). For nuclear staining, cells were incubated for 1 h at RT with 5 µg mL−1 Hoechst 33,258 (Sigma), rinsed three times in PBS, and then mounted. Images were obtained under an ImageXpress® Micro Confocal microscope (Molecular Devices).
10
HSC Proliferation Assay via Hoechst Staining
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HSCs were seeded in black-wall 96-well plates (Corning, cat# 3603) at 3000 cells/well, and 18 hr later, DMSO and NCMC at different concentrations as indicated were added with six replicates. One plate was fixed on each day with 4% paraformaldehyde for five days consecutively and stored at 4 °C until all plates were ready for staining with Hoechst. ImageXpress Micro Confocal microscope (Molecular Devices) was used for taking four images/well with 10 x Plan Apo lens, and MetaXpress software was used for counting the number of nuclei.
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