Heparin was tagged with AF555 during the PDPH modification. A standard curve of heparin fluorescence (Figure S4A –B ) was produced by making serial dilutions of the heparin dissolved in PBS. 150μL of each solution was placed in a 96 well Greiner SensoPlate and imaged using an ImageXpress MicroConfocal Imaging System (Molecular Devices). A 5 mm biopsy punch of each macrogel containing heparin was placed into a well of a 96 well Greiner SensoPlate and covered with 100μL of PBS. Using the same focus and exposure time as the standard curve the macrogel is acquired. Using a custom ImageXpress module (Molecular Devices) the average intensities for each site of the macrogel are calculated (Figure S4C ). The 4 sites with the smallest standard deviation are averaged and the concentration is calculated based on the standard curve (Figure S4D ).
Imagexpress micro confocal imaging system
Manufactured by Molecular Devices
Sourced in United States
The ImageXpress Micro Confocal Imaging System is a high-performance imaging platform designed for automated, high-content screening and imaging applications. The system features a confocal optical design that provides high-resolution, multi-dimensional images with improved signal-to-noise ratio and reduced background fluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Duolink in situ pla kit, by Merck Group (1 mentions)
Fv1000 confocal laser scanning biological microscope, by Olympus (1 mentions)
Metamorph microscopy software, by Molecular Devices (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Anti gm130, by Abcam (1 mentions)
Facsaria 3, by BD (1 mentions)
5 protocols using imagexpress micro confocal imaging system
1
Quantifying Heparin Incorporation in Macrogels
2
Visualizing Mitochondrial-ER Interactions
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In situ proximity ligation assay was performed according to the instructions for the Duolink in situ PLA kit (Sigma-Aldrich). Anti-VDAC1, anti-IP3R1, or anti-GRP75 antibodies were used to visualize MAM formation. Fluorescent blobs were detected using a FV1000 confocal laser scanning biological microscope (Olympus, Tokyo, Japan) or an ImageXpress Micro Confocal Imaging System (Molecular Devices). The number of detected blobs per nucleus was calculated by ImageJ software or MetaMorph Microscopy Software (Molecular Devices).
3
Quantitative Analysis of NTSR1 Internalization
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Adherent cells were rinsed twice with PBS-CM (1X, thermofisher, 14040133), fixed with PFA (4%, PBS-CM 1X) 10 min at room temperature and permeabilized with saponin (0,1%) containing Permeabilization buffer (1X, eBioscience, 00-8333) 10 min at room temperature. Samples were incubated with primary antibodies diluted in 10% FBS, PBS (1X) overnight at 4 °C, then washed with PBS and incubated with secondary antibodies diluted in 10% FBS, PBS (1X), 1 h at room temperature. The following primary antibodies, secondary antibodies and staining molecules were used at the corresponding dilution: anti-NTSR1 (1/100e sigma, SAB43000718), anti-GM130(1/200e, Abcam, ab52649), anti-rabbit Cy3, Phalloidin Alexa fluor 594 (1:200e, thermofisher, A12381), Hoechst (thermofisher, 33258).
The images were acquired with the ImageXpress Micro Confocal Imaging system (Molecular Devices) and quantified with imageJ. For NTSR1 internalization quantification, NTSR1-GFP was measured at the cell periphery (20-pixel wide delimitation around the phalloidin staining) and cell’s interior NTSR1-GFP intensity (. Each NTSR1-GFP cell interior intensity was normalized to its respective cell periphery NTSR1-GFP. Phalloidin intensity measurement was used as control.
The images were acquired with the ImageXpress Micro Confocal Imaging system (Molecular Devices) and quantified with imageJ. For NTSR1 internalization quantification, NTSR1-GFP was measured at the cell periphery (20-pixel wide delimitation around the phalloidin staining) and cell’s interior NTSR1-GFP intensity (. Each NTSR1-GFP cell interior intensity was normalized to its respective cell periphery NTSR1-GFP. Phalloidin intensity measurement was used as control.
4
Visualizing EHF Knockdown in GEA Cells
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GEA cells were stably transfected with a pLVX-GFP-H2B lentiviral vector and then transfected with siEHF or siNC. Quintuplicate wells for each group with six fields per well were photographed every 10 min for 48 h with an ImageXpress Micro Confocal imaging system (Molecular Devices, USA).
5
Sorting SIX6-GFP/VSX2-tdTomato Organoid Cells
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Day 45 SIX6-GFP/VSX2-tdTomato organoids were screened for GFP and tdTomato fluorescence using an ImageXpress Micro Confocal Imaging System (Molecular Devices, San Jose, CA, USA). Organoids were pooled (n = 10–20) and dissociated into single cell suspensions. Organoids were incubated in Accutase for 5 min, swirled gently and incubated for another 5 min. Organoids were washed 3 times in PBS (Ca2+/Mg2+ free), gently dissociated in sort buffer (Ca2+/Mg2+ free PBS, 1mM EDTA, 25mM HEPES pH 7.0, 1% FBS) and filtered (40 µm pore) for a final concentration of 5 × 106 cells/mL. Cells were sorted at the Flow Cytometry Core Facility at the La Jolla Institute for Immunology on a FACSAria-3 (BD Biosciences, Franklin Lakes, NJ, USA).
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