Reagents used are as follows. PB 0.2 M: made by mixing 0.2 M NaH2PO4.1H2O and 0.2 M Na2PO4.2H2O in 19:81 ratio and dilute as needed; 2.3 M sucrose: Sucrose D(+) saccharose in 0.1 M PB; UA, pH 7: dissolve 4 g UA in 100 ml H2O, dissolve 3.8 g oxalic acid in 100 ml H2O, mix 1:1, add NH4OH until pH 7.0 is reached (use pH indicator sticks), and filter at 0.45 µm before use; MC/UA, pH 4: dissolve 0.4 g UA in 10 ml H2O and mix 1:9 with 2% cellulose in H2O; MC: 1.8% MC (25 centipoises, Sigma M-6385) in H2O; gelatin: for 12%, add 12 g food-grade gelatin to 75 ml of 0.1 M PB, warm to 60°C, stir, add 100 µl of 20% Na-Azide, add 0.1 M PB up to 100 ml, and dilute in 0.1 M PB as needed; FA: for 16% stock aliquots, add 80 g PFA (prilled, 441244; Sigma-Aldrich) to 400 ml H2O, warm to 60°C. stir for 15 min, add 0.1 M NaOH until pH is 7 (use indicator sticks), stir for 30 min at 60°C, cool to RT, check pH again, add H2O up to 500 ml, filter solution, freeze aliquots, thaw for use (sometimes heating is required), and do not use if solution does not turn clear; BSA (fraction V, A-9647; Sigma-Aldrich): dilute in H2O; Protein-A gold (protein-A conjugated to colloidal gold particles): made in-house (Cell Microscopy Core, UMC Utrecht) and available online.
M6385
Manufactured by Merck Group
Sourced in Germany
The M6385 is a laboratory equipment product from Merck Group. It is designed for general laboratory use, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information is not available.
Automatically generated - may contain errors
Lab products found in correlation
G5882, by Merck Group (2 mentions)
Oxalic acid, by Merck Group (2 mentions)
Morada tem ccd camera, by Olympus (2 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
A9647, by Merck Group (1 mentions)
Mowiol, by Merck Group (1 mentions)
Dmra fluorescence microscope, by Leica (1 mentions)
Collagen 1 coated, by Thermo Fisher Scientific (1 mentions)
Matrigel coated, by BD (1 mentions)
Click it edu imaging kit, by Thermo Fisher Scientific (1 mentions)
Whatman no 1 filter paper, by Cytiva (1 mentions)
Gatan 781 camera, by Ametek (1 mentions)
Cellvue nir780, by Thermo Fisher Scientific (1 mentions)
Rat tail collagen type 1 solution, by BD (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
P6148, by Merck Group (1 mentions)
Jem 1400 transmission electron microscope, by JEOL (1 mentions)
Glutaraldehyde, by Polysciences (1 mentions)
Fei tecnai t12 electron microscope, by Thermo Fisher Scientific (1 mentions)
Tecnai 20 200 kv lab6tem, by Thermo Fisher Scientific (1 mentions)
13 protocols using m6385
1
Preparation of Buffered Solutions for Microscopy
2
Measuring E98 Cell Proliferation and Migration
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E98 cells were grown on collagen I-coated (10 μg/cm2; Invitrogen) coverslips to 60–80 % confluency over 48 h, and incubated for 1 h with culture medium containing 10 μM EdU (5-ethynyl-2′-deoxyuridine). EdU incorporation was visualized using the click-iT® EdU Imaging kit (Thermo Fisher Scientific, #C10086) via the manufacturers’ instruction. Coverslips were mounted on microscope slides in DAPI-containing Mowiol (Sigma-Aldrich) and images were collected on a Leica DMRA Fluorescence microscope, equipped with a DFC340 FX CCD camera, using 40x and 63x objectives. DAPI- and EdU-positive nuclei were counted automatically using FIJI software [28 (link)].
Migration of E98 cells was assessed in spheroid outgrowth assays as follows. E98 spheroids were generated in hanging drops using methylcellulose (12 mg/mL; Sigma, M6385) in DMEM supplemented with 10 % FCS (2500 cells per spheroid). The next day, individual spheroids were seeded in a 96-well imaging culture dish (BD Falcon, #353219) on top of a confluent mouse astrocyte layer in Matrigel-coated (30 μg/mL PBS; BD Biosciences, #356237) culture wells. 24 h later, cells were fixed and fluorescent (tagRFP) images were collected. Average migration distance of cells from spheroids (n > 37), calculated as change in radius of the spheroid over 24 h, were analyzed semi-automatically using FIJI software.
Migration of E98 cells was assessed in spheroid outgrowth assays as follows. E98 spheroids were generated in hanging drops using methylcellulose (12 mg/mL; Sigma, M6385) in DMEM supplemented with 10 % FCS (2500 cells per spheroid). The next day, individual spheroids were seeded in a 96-well imaging culture dish (BD Falcon, #353219) on top of a confluent mouse astrocyte layer in Matrigel-coated (30 μg/mL PBS; BD Biosciences, #356237) culture wells. 24 h later, cells were fixed and fluorescent (tagRFP) images were collected. Average migration distance of cells from spheroids (n > 37), calculated as change in radius of the spheroid over 24 h, were analyzed semi-automatically using FIJI software.
3
Wheat Seed Biocontrol Treatment Protocol
4
Electron Microscopy of (V)LDL Subfractions
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Both the (V)LDL precipitate (resuspended in 1× PBS) and its sub-fractions from density gradient centrifugation (100 µL leftover of each sub-fraction) were processed at room temperature (20 °C) for electron microscopy (EM). The samples were diluted 1:5 in 1× PBS prior to fixation with 2.0% glutaraldehyde (Sigma-Aldrich #G5882, St. Louis, MO, USA). After fixation, a 75-mesh grid (Agar scientific #G2075C, Stansted, UK) was laid on a drop of sample for 10 min; then, the grid was rinsed 10 times with Milli-Q H2O (1 min per rinse). For staining, the grid was firstly laid on a drop of uranyl acetate (pH 7.0, SPI-CHEM #2624, West Chester, PA, USA) for 10 min. The grid was subsequently rinsed with Milli-Q H2O and methylcellulose uranyl (pH 4.0), and then incubated for 10 min on a drop of methylcellulose uranyl (pH 4.0, Sigma-Aldrich #M-6385, St. Louis, MO, USA). The samples were analyzed with an FEI Tecnai™ T12 electron microscope (FEI Company, Eindhoven, The Netherlands).
5
Exosome Characterization by Electron Microscopy
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Frozen, isolated exosomes were resuspended in 1% buffered formaldehyde, adsorbed onto formvar-carbon-coated electron microscopy grids (EMS, FCF200H-Cu), and fixed with a mixture of 2% formaldehyde for 20 min. A 50-µL drop of PBS was placed on a sheet of parafilm, and grids were transferred with the sample side facing down on the drop for 2 min. The grids were transferred to a 50-µL drop of 1% glutaraldehyde for 5 min prior to transfer to a 100-µL drop of distilled water for 2 min. Negative staining was performed by placing the grid over a 50-µL drop of uranyl-oxalate solution for 5 min (4% uranyl acetate, EMS, 22400-4; 0.15 M oxalic acid, Sigma-Aldrich, 75688 pH 7) before transfer to a 50-µL drop of methyl-cellulose-UA (4% uranyl acetate and 2% methyl cellulose for 10 min, Sigma-Aldrich, M6385, in a ratio of 100 µL:900 µL) on a glass dish covered with parafilm on ice. The grids were removed, and excess fluid was blotted gently onto Whatman no.1 filter paper. The grids were dried, stored in appropriate grid storage boxes, and subsequently observed under a JEOL 1200 EX II transmission electron microscope at 80 kV. Images were acquired with a GATAN 781 camera (USA).
6
Negative Staining Transmission Electron Microscopy of Viral Particles
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Viral particles were visualized by NS-TEM, as reported previously (Notaro et al. 2021 (link)). Briefly, viral particles were fixed in glutaraldehyde (2.5% v/v in water) for 1 h at room temperature. Samples were centrifuged at 5000 g for 10 min and pellets were washed twice with water. The structure of the fibrils was visualized by NS-TEM using methyl cellulose (M6385 Sigma) and uranyl acetate (2% v/v in water) as reported (Notaro et al. 2021 (link)). Viral particles were observed by TEM on a TECNAI G2°200 KV.
7
Sprouting Angiogenesis in LEC Spheroids
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Spheroids of LEC were generated from 2000 cells in hanging drops in ECGM2 containing 20% of methylcellulose (M6385, Sigma Aldrich). As previously described in ref. 56 , spheroids were embedded in a collagen gel (08-115, Merck, Germany) and cultured in the respective medium of LEC. Depending on experimental settings, cells were stimulated with 100 ng/mL VEGF-C, treated with 25 µM CQ, 20 mM ammonium chloride (NH4Cl), and 100 µM leupeptin or supplemented with 20 mM AC and cultured for 48 h to analyze sprouting. For the mixed spheroids, LEC were collected 48 h after transfection and labeled with cell membrane labeling lipophilic dyes CellVue™ Jade and CellVue™ NIR780 (88-0876-16 and 88-0875-16 respectively, Thermo Fisher Scientific). Spheroids containing equal amounts of si CTRL transfected cells (green) and si ATG5 transfected cells were generated, embedded, and analyzed after 48 h. Images were taken in an inverted microscope (IX83, Olympus) and an analysis for number of sprouts per spheroid was performed using NIH ImageJ software.
8
3D Spheroid Collagen Invasion Assay
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Multicellular spheroids were obtained from sub-confluent cells using the hanging-drop technique (2% methylcellulose, cod. M6385, Sigma Aldrich, Merck, Darmstadt, Germany; 500 cells/50 L drop; 7 days spheroid assembly) according to Ref. [44 (link)]. Spheroids were embedded in non-pepsinzed rat-tail collagen type I solution (2.5 mg/mL; BD Biosciences, Franklin Lakes, NJ, USA) prior to collagen polymerization (37 C for 5–10 min). The collagen matrix invasion process was monitored by bright-field time-lapse microscopy using a LEICA DMI8 microscope (Leica, Germany) acquiring one frame per hour for 72 h. The image analysis is done using Matlab Image Processing Toolbox. At first, the image is cut to focus on the area of interest. Then it is thresholded and a black-white image is created using built-in Matlab function “edge”. After it is dilated with “imdilate” and the holes are filled with “imfill”. Small noise is removed by “bwareaopen”. Then the image is eroded and filled by “imerode” and “imfill” functions. In the resulting images the center of the white (detected) area is found. Then, we compute the average distance from the center to all other detected points. This value corresponds to the spread at a given time step.
9
Transmission Electron Microscopy of Extracellular Vesicles
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Suspension of EVs was deposited on formvar-carbon-coated 200 mesh cooper grids (Agar Scientific, Essex, UK) for TEM analysis according to the method described by Thery et al. 200,615. Briefly, EVs on grids were fixed in 2% paraformaldehyde (P6148, Sigma-Aldrich, Schnelldorf, Germany) and 1% glutaraldehyde (O 1909–10, Polysciences, Warrington, USA), before being contrasted in uranyl oxalate [mixture of 4% uranyl acetate (21447–25, Polysciences, Warrington, USA) and 0,15 M oxalic acid (75,688, Sigma-Aldrich, Schnelldorf, Germany)] and embedded in a mixture of methylcellulose (M6385, Sigma-Aldrich, Schnelldorf, Germany) and uranyl acetate (21447–25, Polysciences, Warrington, USA). Samples were observed with a JEM 1400 transmission electron microscope (JEOL Ltd. Tokyo, Japan) at 80 kV, and digital images were acquired with a numeric camera (Morada TEM CCD camera, Olympus, Germany).
10
Transmission Electron Microscopy of Exosomes
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The isolated exosomes were processed at room temperature for TEM. The samples were diluted 1:5 in 1X PBS prior to fixation with 2.0 % glutaraldehyde (G5882, Sigma-Aldrich). Following fixation, a 75-mesh grid (G2075C; Agar Scientific, Essex, UK) was laid on a drop of sample for 10 min; the grid was then rinsed 10 times with MiliQ H2O (1 min per rinse). Subsequently, the grid was firstly laid on a drop of uranyl acetate (pH 7.0, 2624; SPI-CHEM, West Chester, PA, USA) for 10 min. After rinsing with Milli-Q H2O and methylcellulose uranyl (pH 4.0), the grid was incubated at room temperature for 10 min on a drop of methylcellulose uranyl (pH 4.0, M-6385, Sigma-Aldrich). The exosome samples were eventually analyzed with a FEI Tecnai™ T12 electron microscope (Thermo Fisher Scientific).
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