Samples for imaging were set up in 16-well CultureWells (Grace BioLabs). Wells were passivated by overnight incubation in 5% (wt/vol) Pluronic acid (Thermo Fisher), and washed thoroughly with the corresponding buffer prior to use. All condensate samples were incubated for 30 min prior to imaging unless otherwise stated. For experiments where samples were imaged across pH titrations, the following buffers were used: phosphate buffer for pH 6.3–6.7, HEPES for pH 7.2–7.7, and Tris–HCl for 8.2–8.6. Imaging of condensates was performed using a Nikon Ti2-E motorized inverted microscope (60× DIC objective and DIC analyzer cube) controlled by NIS Elements software with a Transmitted LED Lamp house and a Photometrics Prime 95B Back-illuminated sCMOS Camera. Image analysis was performed using Fiji v 1.0.
Culturewell
Manufactured by Grace Bio-Labs
Sourced in United States
CultureWell is a versatile laboratory equipment designed for cell culture applications. It provides a controlled environment for the growth and maintenance of cells, ensuring optimal conditions for cell proliferation and viability.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (4 mentions)
Pluronic acid, by Thermo Fisher Scientific (3 mentions)
Hexidium iodide, by Thermo Fisher Scientific (2 mentions)
Ti2 e motorized inverted microscope, by Nikon (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Hank s balanced salt solution, by Lonza (1 mentions)
Dmi6000b fluorescence microscope, by Leica (1 mentions)
Thunder microscope, by Leica (1 mentions)
Twomp mass photometer, by Refeyn (1 mentions)
A1rsi, by Nikon (1 mentions)
10 protocols using culturewell
1
Imaging Condensates: Standardized Setup
2
SUMO-tagged Protein Cleavage Visualization
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Prior to imaging, His–SUMO–McdB samples from S. elongatus 7942 were buffer exchanged into a Ulp1 reaction buffer (150 mM KCl; 25 mM HEPES; 2 mM BME) and adjusted to the indicated pH. Similarly, His–SUMO–McdB samples from each of the Type 2 systems chosen were buffer exchanged into a defined Ulp1 reaction buffer, being Gloeobacter kilaueensis JSI (150 mM NaCl; 25 mM HEPES, pH 7.0; 2 mM BME), Fremyella diplosiphon NIES-3275 (25 mM HEPES, pH 7.0; 2 mM BME), and Fischerella sp. PCC 9431 (150 mM NaCl; 25 mM HEPES, pH 7.0; 2 mM BME). Buffer exchange was performed using 7 K MWCO, 5 ml Zeba Spin Desalting Columns (Thermo-Fischer). All imaging was performed using 16 well CultureWells (Grace BioLabs). Wells were passivated by overnight incubation in 5% (w/v) Pluronic acid (Thermo-Fischer), and washed thoroughly with the corresponding Ulp1 buffer prior to use. For cleavage experiments, 1 µl of purified Ulp1 was added to 50 µl of His–SUMO–McdB at the indicated concentration and incubated at 23 °C for 2 h to ensure complete cleavage. Imaging of McdB droplet formation was performed using a Nikon Ti2-E motorized inverted microscope (60× DIC objective and DIC analyzer cube) with a Transmitted LED Lamp house and a Photometrics Prime 95B Back-illuminated sCMOS Camera. Image analysis was performed using Fiji v 1.0.
3
Optimal Transfection of TIB73 Cells
4
Imaging of McdB Droplet Formation
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Images were taken of 450 µM McdB in a buffer consisting of 20 mM HEPES (pH 7.0) and 100 mM KCl, with or without the addition of either 15% (w/v) PEG‐8000 or Ficoll‐400 as indicated. All samples were left to incubate for 2 hr prior to imaging at room temperature. Imaging was performed using 16 well CultureWells (Grace BioLabs). Wells were passivated by overnight incubation in 5% (w/v) Pluronic acid (Thermo‐Fischer), and washed thoroughly with the corresponding buffer prior to use. Imaging of McdB droplet formation was performed using a Nikon Ti2‐E motorized inverted microscope (60 × DIC objective and DIC analyzer cube) with a Transmitted LED Lamp house and a Photometrics Prime 95B Back‐illuminated sCMOS Camera. Image and video analyses were performed using Fiji v 1.0.
5
Bacterial Growth and Biofilm Analysis
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Bacterial growth and biofilm formation were analyzed using confocal laser scanning microscopy, as described previously14 (link), 49 (link), with some modifications. In the bacterial growth assay, 500 μL of the cultured bacteria was collected by centrifugation, resuspended in 500 μL of PBS including 2.5 µl of 10 mM hexidium iodide (Invitrogen, Carlsbad, CA, USA), and incubated in the dark for 15 min at room temperature. Then, 20 μL of the bacterial suspension applied to the cover glass was fixed with 3% paraformaldehyde (FUJIFULM Wako Pure Chemical Corporation). For the biofilm assay, the biofilm formed on a chambered cover glass system (CultureWell™, Grace Bio Labs, Bend, OR, USA) or S. mutans cultured on the enamel test piece was stained with 5 µl of 10 mM hexidium iodide in 1 ml of Hanks’ balanced salt solution (Lonza, Walkersville, MD, USA) for 15 min at room temperature in the dark. The plates were then washed with PBS and fixed with 3% paraformaldehyde. Bacterial growth and biofilm formation were observed by confocal scanning laser microscopy using a LSM510 (Carl Zeiss, Oberkochem, Germany) with reflected laser light at 543 nm, as well as a DMI6000 B fluorescence microscope (Leica Microsystems GmbH) and a 63 × oil immersion objective.
6
Visualizing Kinesin-1 Movement on MTs
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Cy5-labeled MTs were polymerized in the presence of GTP and phase-separated CPC. Cy-5 tubulin was from PurSolutions. Reaction mixture was transferred into coverslip-bottomed CultureWell (Grace BioLabs), and then solution of glucose/oxidase system (40 mM glucose, 130 mg/ml glucose oxidase, and 24 mg/ml catalase), 1 mM ATP, and 30 nM of kinesin-1-GFP was added. Kinesin movements were imaged using 100× objective in a TIRF mode with excitation at 480 nm on the LEICA Thunder microscope. Images were taken every 220 ms. Analysis was done in FIJI (National Institutes of Health and the Laboratory for Optical and Computational Instrumentation (LOCI, University of Wisconsin)).
7
Mass Photometry Characterization of Protein Binding
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Data were taken using a TwoMP mass photometer (Refeyn,
UK) and analyzed using a custom-written Python package, based on the
procedure described in Young et al.3 (link) Coverslips
(Menzel-Gläser, 24 × 50 mm # 1.5 SPEZIAL; Thermo Fisher
Scientific, U.S.) were cleaned, a silicon gasket (Grace Bio-Labs CultureWell,
3 × 1 mm; U.S.) was laid on top, and 4 μL of buffer medium
was added. Next, the protein was diluted in an Eppendorf tube (Eppendorf,
1.5 mL; Germany) to give 20 μL of sample and added into the
gasket. Movies containing ∼1000–5000 binding events
were recorded (60 s), analyzed, and converted into mass using a calibration
curve (generated from a measurement of the oligomeric peaks of dynamin-1
ΔPRD).
SUMO-SRSF1 was diluted to 20 nM in a buffer of
20 mM HEPES (pH 7.4), 1 M NaCl, 1 mM DT, and 20 mM NaCl. A 20 mM Tris
(pH 7.4) and 50 mM NaCl buffer was used to dilute Starmaker, allowing
for a reduction or an increase in salt concentration upon measurement.
For both proteins, 4 repeats were taken at each condition, with no
significant unbinding in any repeat.
UK) and analyzed using a custom-written Python package, based on the
procedure described in Young et al.3 (link) Coverslips
(Menzel-Gläser, 24 × 50 mm # 1.5 SPEZIAL; Thermo Fisher
Scientific, U.S.) were cleaned, a silicon gasket (Grace Bio-Labs CultureWell,
3 × 1 mm; U.S.) was laid on top, and 4 μL of buffer medium
was added. Next, the protein was diluted in an Eppendorf tube (Eppendorf,
1.5 mL; Germany) to give 20 μL of sample and added into the
gasket. Movies containing ∼1000–5000 binding events
were recorded (60 s), analyzed, and converted into mass using a calibration
curve (generated from a measurement of the oligomeric peaks of dynamin-1
ΔPRD).
SUMO-SRSF1 was diluted to 20 nM in a buffer of
20 mM HEPES (pH 7.4), 1 M NaCl, 1 mM DT, and 20 mM NaCl. A 20 mM Tris
(pH 7.4) and 50 mM NaCl buffer was used to dilute Starmaker, allowing
for a reduction or an increase in salt concentration upon measurement.
For both proteins, 4 repeats were taken at each condition, with no
significant unbinding in any repeat.
8
Fabrication of Fano Resonant Plasmonic Metasurfaces
9
Streptococcus mutans Adhesion Assay
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Streptococcus mutans binding to type IV collagen and fibrinogen was also assessed using confocal laser scanning microscopy as described previously52 (link) with some modifications. Type IV collagen or fibrinogen were added to chambered coverglass wells (CultureWell; Grace Bio Labs, Bend, OR, USA) and incubated overnight at 4 °C. The coated wells were washed three times with PBS, blocked for 1.5 h with 5% BSA in PBS at 37 °C, and washed again with PBS containing 0.01% Tween 20. S. mutans cells were collected, stained with hexidium iodide (Molecular Probes), and added to the coated wells (2 × 109 CFU/well) in PBS. The cells were cultured anaerobically at 37 °C for 18 h in the dark. Non-attached S. mutans cells were removed by washing with PBS, and the adherent cells were observed using a confocal laser scanning microscope (LSM510; Carl Zeiss, Oberkochem, Germany) with a 63 × oil immersion objective.
10
3D Imaging of Self-Assembled Gel Microstructures
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An inverted confocal laser scanning microscope (CLSM) (Nikon A1Rsi, equipped with NA = 1.4, 100x objective, oil-immersion type) is used to image the 3D microstructures of the gels selfassembled from fluorescently labeled particles. After the addition of MgCl2 solution, the suspension is briefly and gently mixed for homogeneity and loaded into a 16-well chambered cover glass (Grace Bio-Labs, CultureWell, ChamberSLIP 16) mounted on the microscope stage above the objective. The chamber was closed to prevent evaporation. The gels form quiescently for 45 minutes before imaging. For visualization and microstructure characterization, 3D image volumes of size 512 x 512 pixels with pixel size 0.083 µm were acquired, beginning at the coverslip. The image stacks comprised of ~ 200 slices spaced at 0.083µm (acquired using Nikon AI Piezo z-drive). While acquiring image volumes, the intensity gain was gradually increased in the z-direction in steps of 1 unit for every 0.5 µm to compensate for the loss of image intensity at depths greater than 8 µm due to the refractive index mismatch between polystyrene particles and H2O-D2O solution. CLSM visualization of the purified discoids are observed to be free of selfaggregation prior to the start of gelation (Supplementary Figure 4).
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