The photophysical properties of photoconversion have been characterized using a paste of bacterial cells. About 2 μl of bacterial paste expressing the miRFP protein of interest was placed on a #1.5 coverslip and compressed on a slide, to obtain a homogeneous layer of bacteria, and finally sealed with biphasic glue. To probe the influence of oxygen on photoconversion, this mounting approach (referred to as “closed-chamber” in Supplementary Fig. 20 ) was compared to a similar amount of bacteria paste placed on an open chamber (MatTek, P35G-1.5-20-C) with a #1.5 coverslip bottom. Plasmids used for bacterial expression of the green-to-orange PCFPs were purchased from Addgene and are the following: pGEX6P-1-Dendra2 (Plasmid # 82436).
P35g 1.5 20 c
Manufactured by MatTek
Sourced in United States
The P35G-1.5-20-C is a cell culture dish designed for microscopy applications. It features a glass bottom with a thickness of 1.5 mm and a diameter of 35 mm. The dish has a growth area of 20 cm^2.
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Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (6 mentions)
P35g 1.5 14 c, by MatTek (4 mentions)
Streptomycin, by Merck Group (4 mentions)
Penicillin, by Merck Group (4 mentions)
Volocity software, by PerkinElmer (4 mentions)
Ultraview vox spinning disk confocal system, by PerkinElmer (4 mentions)
Alexa fluor 488 goat anti rabbit secondary, by Thermo Fisher Scientific (4 mentions)
Csu x1 spinning disk head, by Hamamatsu Photonics (4 mentions)
C9100 13, by Nikon (4 mentions)
Alexa fluor 568 goat anti mouse secondary, by Thermo Fisher Scientific (4 mentions)
Rr060058, by Thermo Fisher Scientific (3 mentions)
D glucose, by Merck Group (3 mentions)
Neurobasal, by Thermo Fisher Scientific (3 mentions)
D1306, by Thermo Fisher Scientific (3 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (3 mentions)
Hc pl apo cs oil objective, by Leica (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Matlab, by MathWorks (2 mentions)
Lida light engine, by Lumencor (2 mentions)
Prime 95b camera, by Teledyne (2 mentions)
59 protocols using p35g 1.5 20 c
1
Characterizing Photoconversion Properties
2
Simultaneous BODIPY-Cer Exocytosis Monitoring
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Cells were cultured in glass-bottom dishes (P35G-1.5-20-C; Mattek) and loaded with BODIPY-Cer (B22650; Invitrogen; 5 µM) by incubation at 4°C for 15 min. Cells were washed with 37°C PBS and immediately transferred to the microscope stage. For co-exocytosis assays, cells were transfected with EQ-SM-mKate2 and EQ-sol-mKate2. BODIPY-Cer exocytosis events were counted manually according to experimental limitations. For co-exocytosis assays, EQ-SM-mKate2 or EQ-sol-mKate2 fusion events were used to search for co-fusion events with BODIPY signals at the same region and time.
3
Pacifistic Sphingosine Tracing in Cells
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The experiments were performed as described previously (Sundberg et al., 2019a (link)). Briefly, 1 million SGPL1 null cells were seeded into glass-bottom dishes (P35G-1.5-20-C; Mattek) and experiments were initiated within 16 h after plating. Cells were pulse-labeled for 15 min with 1 μM pacSphingosine (pulse) and chased for the indicated time period. Cells were fixed in 4% PFA in PBS (15710; Electron Microscopy Sciences) for 15 min and permeabilized with 0.05% saponin for 5 min (control sample for non-labeling). After processing cells for immunofluorescence with antisera to the indicated proteins, cells were incubated with click labeling reagents (C10269; Invitrogen) and Alexa Fluor 647 azide (A10277; Invitrogen) according to the manufacturer’s protocols.
4
Fluorescent Labeling and Phase Separation of Synucleins
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Wild-type α-synuclein and β-synuclein were purified from Escherichia coli expressing plasmid pT7-7 encoding for the protein as previously described (38 (link), 39 ). Following purification, the protein was concentrated using Amicon Ultra-15 centrifugal filter units (Merck Millipore) and buffer exchanged into phosphate-buffered saline (PBS) at pH 8.0. Protein was subsequently labeled with 10-fold molar excess of fluorescein 5-isothiocyanate (Sigma) for 3 h at room temperature, followed by an overnight incubation at 4 °C with constant mixing. The excess dye was removed on a Sephadex G-25 desalting column (Sigma) and used immediately for phase separation experiments.
To induce droplet formation, nonlabeled wild-type α-synuclein and β-synuclein were mixed with FITC-labeled proteins at a 10:1 molar ratio in PBS with 50 mM NaCl and 10% polyethylene glycol (PEG) (Thermo Fisher Scientific). The final mixture was pipetted onto a 35-mm glass-bottom dish (P35G-1.5-20-C; MatTek Life Sciences) and immediately imaged on a TCS SP5 confocal microscope using a 40×/1.3 HC PL Apo CS oil objective (Leica Microsystems) with the temperature controlled at either 20 or 30 °C. The excitation wavelength was 488 nm for all experiments. All images were processed and analyzed in ImageJ (NIH).
To induce droplet formation, nonlabeled wild-type α-synuclein and β-synuclein were mixed with FITC-labeled proteins at a 10:1 molar ratio in PBS with 50 mM NaCl and 10% polyethylene glycol (PEG) (Thermo Fisher Scientific). The final mixture was pipetted onto a 35-mm glass-bottom dish (P35G-1.5-20-C; MatTek Life Sciences) and immediately imaged on a TCS SP5 confocal microscope using a 40×/1.3 HC PL Apo CS oil objective (Leica Microsystems) with the temperature controlled at either 20 or 30 °C. The excitation wavelength was 488 nm for all experiments. All images were processed and analyzed in ImageJ (NIH).
5
Single-Molecule Tracking and FRAP Imaging
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For both SMT and FRAP, ~200,000 cells were plated in a 35 mm dish containing either a 20 mm diameter or 14 mm wide uncoated coverslip (MatTek Corporation #P35G-1.5-20-C or #P35G-1.5-14-C) one day prior to imaging. Before imaging cells were labeled with either HaloTag-JF549 or JFX549 dye at 5 nM, for SMT, or 50 nM, for FRAP. After 20 min of incubation, free dye was removed by replacing media with 2 mL of phenol-red free media and incubating for 15 minutes. Following a second media replacement and incubation, the media was replaced a final time and cells were imaged.
6
Intracellular Parasite Cultures for Immunofluorescence
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Cultures of intracellular parasites growing in HFF cells in 3.5-cm glass-bottom dishes (P35G-1.5-20-C; MatTek, Ashland, MA) were incubated at 8°C for 3.5 h in CO2-independent medium (Hu et al., 2006 ; RR060058; Life Technologies, Carlsbad, CA) supplemented with 1% (vol/vol) heat-inactivated cosmic calf serum (CCS; SH30087.3; Hyclone, Logan, UT), GlutaMAX (35050-061; Life Technologies), sodium pyruvate (11360; Life Technologies), and antibiotic/antimycotic (30-004-CI; Corning, Oneonta, NY). The samples were then processed for immunofluorescence as described next.
7
In vitro phase separation assay for N protein
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The in vitro phase separation assays were performed as described previously (21 (link)). Samples of N protein in 10.1 mM Na2PO4, 1.8 mM KH2PO4, 2.7 mM KCl, 10 mM NaCl, 10% and polyethylene glycol (pH 7.40) at room temperature were transferred onto a 35-mm glass-bottom dish (catalog no. part no: P35G-1.5-20-C, MatTek). Condensates from LLPS were imaged within 30 to 40 min or a few hours, as indicated. Images were acquired on a Nikon Ti-E microscope equipped with a 100×1.49 numerical aperture oil objective lens (LIDA light engine, Lumencor, Beaverton, OR) and recorded with a Prime 95B camera (Teledyne Photometrics) with a pixel size of 110 nm. Images were background-subtracted and contrast-enhanced using MATLAB (Mathworks, Natick, MA).
8
Imaging Fission Yeast Cells In Vivo
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Fission yeast cells were grown at 25°C in YE4S medium to logarithmic phase for imaging. Cells were collected at 4,000 rpm for 15 s, placed on a coverglass-bottom dish (P35G-1.5-20C; MatTek), and covered with a piece of YE4S agar prewarmed to 25°C. Images were collected using a spinning disk confocal microscope: Yokogawa CSU-WI (Nikon Software) equipped with a 60× 1.4-NA CFI60 Apochromat Lambda S objective lens (Nikon); 405-, 488-, and 561-nm laser lines; and a photometrics Prime BSI camera on an inverted microscope (Eclipse Ti2; Nikon). Multiple fields per cell type were imaged within 1 h at room temperature, and images were acquired with 27 z-stacks and 0.2-µm steps.
9
Lrrk2-G2019S Cortical Neuron Isolation
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All experiments were performed following protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Lrrk2-p.G2019S KI mice (model #13940) and B6NTac mice (model #B6) were obtained from Taconic, Cambridge City, Indiana production site. Mouse cortices were dissected from homozygous WT or Lrrk2-p.G2019S embryos of either sex at day 15.5. Cortical neurons were isolated by digestion with 0.25% Trypsin and trituration through a small-bore serological pipette. Neurons were plated on 35 mm glass-bottom imaging dishes (P35G-1.5-20-C; MatTek) in Attachment Media (MEM supplemented with 10% horse serum, 33 mM D-glucose and 1 mM sodium pyruvate). After 5 hours, Attachment Media was replaced with Maintenance Media (Neurobasal [GIBCO] supplemented with 2% B-27 [GIBCO], 33 mM D-glucose [Sigma], 2 mM GlutaMAX [GIBCO], 100 U/mL penicillin and 100 mg/mL streptomycin [Sigma]). AraC (1 μM) was added the day after plating to prevent glia cell proliferation. 40% of the media was replaced with fresh Maintenance Media twice per week. Transfections of DIV6 mouse cortical neurons were performed 16-24 hours before imaging using Lipofectamine 2000 Transfection Reagent (Thermo Fisher) and 0.9 μg total plasmid DNA. Published protocol can be found on Protocols.io (https://doi.org/10.17504/protocols.io.81wgby723vpk/v1 ).
10
Lrrk2-G2019S Cortical Neuron Isolation
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