The presence of large insoluble SOD1 aggregates was determined by filter‐trap assays as described before (Wang et al, 2002 ). Briefly, 10 μg of protein samples (S1 and mitochondrial fractions) was treated with 0.5% (v:v) Triton X‐100 for 15 min on ice prior to loading in a Bio‐Dot® Microfiltration apparatus (Bio‐Rad). Cellulose acetate membranes (0.2 μm pore diameter, Whatman Gmbh; Dassel, Germany) were used for the assay, and samples were allowed to pass through the filter by applying vacuum. After several washes, membranes were blocked in 5% (w:v) non‐fat‐dried milk prepared in PBS plus 0.1% Tween‐20 (PBST). Anti‐SOD1 (1:1,000) antibody was used for detection.
Cellulose acetate membrane
Manufactured by Cytiva
Sourced in United Kingdom
Cellulose acetate membrane is a type of laboratory filtration membrane. It is designed for general-purpose filtration applications in various industries, including pharmaceuticals, biotechnology, and environmental analysis. The membrane's primary function is to separate and retain particles, microorganisms, or other suspended solids from liquids during the filtration process.
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Lab products found in correlation
Whatman, by Cytiva (21 mentions)
Bio dot microfiltration apparatus, by Bio-Rad (4 mentions)
Bio dot sf microfiltration apparatus, by Bio-Rad (2 mentions)
Rabbit anti chchd10, by Proteintech (2 mentions)
Rabbit anti chchd2, by Proteintech (2 mentions)
Astra 5, by Wyatt Technology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Lightwave 2, by Harvard Bioscience (1 mentions)
Ab204, by Mettler Toledo (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Dot blotter, by Bio-Rad (1 mentions)
Bio dot device, by Bio-Rad (1 mentions)
Microfuge, by Eppendorf (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Irdye 800 conjugated goat anti rabbit, by LI COR (1 mentions)
21 protocols using cellulose acetate membrane
1
Quantifying Insoluble SOD1 Aggregates
2
Biomass and Cell Size Analysis
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At the end of each 24 h step of light increment, 22 ml of culture were harvested to perform in parallel dry weight measurements and cell size analysis.
For the determination of dry cell weight, cellulose acetate membranes (0.2 μm, Whatman, Maidstone, United Kingdom) were washed with milli-Q water (Merck Millipore Reference, Burlington, MA, United States), left to dry for 24 h at 90 °C in a stove (Electrolux, Stockholm, Sweden) and weighted with an analytical balance (AB204; Mettler Toledo, Columbus, OH, United States). Subsequently, the membranes were used for filtering 20 ml of sampled culture. After washing once with milli-Q water to remove salts, the membrane filter was left to dry overnight in the stove at 90 °C and finally weighted again. In parallel, the OD730 of the sampled cells was measured with a spectrophotometer (Lightwave II; Biochrom, Cambridge, United Kingdom) and used to normalize the dry cell weight per OD730.
The average cell size and cell number were measured with the CASY counter instrument (Roche Applied Science, Penzberg, Germany). A volume of 20 μl of harvested culture was diluted with 10 ml of CASY ton solution. The average cell size was measured working in a range of calibration between zero and five μm with a capillary of 60 μm.
For the determination of dry cell weight, cellulose acetate membranes (0.2 μm, Whatman, Maidstone, United Kingdom) were washed with milli-Q water (Merck Millipore Reference, Burlington, MA, United States), left to dry for 24 h at 90 °C in a stove (Electrolux, Stockholm, Sweden) and weighted with an analytical balance (AB204; Mettler Toledo, Columbus, OH, United States). Subsequently, the membranes were used for filtering 20 ml of sampled culture. After washing once with milli-Q water to remove salts, the membrane filter was left to dry overnight in the stove at 90 °C and finally weighted again. In parallel, the OD730 of the sampled cells was measured with a spectrophotometer (Lightwave II; Biochrom, Cambridge, United Kingdom) and used to normalize the dry cell weight per OD730.
The average cell size and cell number were measured with the CASY counter instrument (Roche Applied Science, Penzberg, Germany). A volume of 20 μl of harvested culture was diluted with 10 ml of CASY ton solution. The average cell size was measured working in a range of calibration between zero and five μm with a capillary of 60 μm.
3
Measuring Dry Cell Mass in Cyanobacteria
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Dry cell mass was determined as previously described with some modifications (68 (link)). S. elongatus cultures (∼45 mL) were harvested after 24 h postinduction by centrifugation at 4,000 rpm for 30 min. Pellets were washed twice with distillated water and transferred onto cellulose acetate membranes (0.45 μm; Whatman) and immediately dried in a stove for ≥4 h at 90 °C. The mass of each membrane was measured with an analytical balance before and after adding the cells, and these data were used to calculate the dry cell weight per volume.
4
Scalable Secretome Generation Protocol
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To generate secretomes, 3×106 sensitive cells and 7×106 DR clones were plated on 15 cm plates in standard media and allowed to adhere overnight. The media was then replaced with fresh media with 2% dialyzed FBS and indicated drugs for 48 hr. FBS was dialyzed in-house (against 0.15 M NaCl until glucose reached <5 mg/dL) using 10 k MWCO dialysis tubing (Fisher Scientific) at 4 °C for 6 hr. Secretomes were centrifuged at 1000 r.p.m. for 5 min, vacuum filtered using 0.45 μm cellulose acetate membranes (Whatman), and immediately placed on ice. For 2D co-culture, secretomes were stored at 4 °C, warmed prior to use, and were used only within 48 hr. For 3D co-culture, only freshly prepared secretomes were used and were further concentrated using Amicon Ultra-15 mL Centrifugal Filters (3 k MWCO). For biochemical assays, secretomes were further concentrated using Amicon Ultra-15 mL Centrifugal Filters (3 k, 10 k, 30 k, 50 k, 100 k MWCO as indicated) and depleted of albumins and IgGs using MARS HAS/IgG spin columns. Aliquots were ‘snap-frozen’ in liquid nitrogen and stored at −80 °C until use. Aliquots were thawed only once.
5
Quantifying Protein Aggregation in NSC-34 Cells
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NSC-34 cells were plated at 80,000 cells/well in 12-well plates, transfected and collected 48h after transfection in PBS added with protease inhibitor. Cells were homogenized using slight sonication to lyse cells and nuclei as previously described (29 (link)). Filter retardation assay (FRA) was performed using a Bio-Dot SF Microfiltration Apparatus (Bio-Rad). 6μg of the total protein extracts were filtered through 0.22μm cellulose acetate membranes (Whatman, 100404180). The membranes were probed as described for WB. A ChemiDoc XRS System (Bio-Rad) was used for image acquisition. The optical density of samples assayed with WB or FRA was detected and analyzed using the Image Lab software (Bio-Rad). Statistical analyses were performed using relative optical densities, defined as the ratio between the optical density of each independent biological sample (n = 3) and the mean optical density of control samples.
6
Dry Cell Mass Determination in Cyanobacteria
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Dry cell mass was determined as previously described [26 (link)]. S. elongatus cultures (~47.5 mL) were harvested after 24 hr post-induction by centrifugation at 4,000 rpm for 30 min. Pellets were washed twice with distillated water and transferred onto cellulose acetate membranes (0.45 μm, Whatman) and immediately dried in a hot air oven for ≥4 hr at 90°C. The mass of each membrane was measured with an analytical balance before and after adding the cells, and these data were used to calculate the dry cell weight per volume.
7
Determination of Bacterial Dry Cell Mass
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Dry cell
mass was determined as previously described.64 (link)E. coli cultures (20 mL) were harvested
hourly during the growth curve by centrifugation at 6000g for 30 min. Pellets were washed twice with distilled water and transferred
onto cellulose acetate membranes (0.45 μm, Whatman) and immediately
dried in a hot air oven for ≥4 h at 90 °C. The mass of
each membrane was measured with an analytical balance before and after
adding the cells, and these data were used to calculate the dry cell
weight per volume.
mass was determined as previously described.64 (link)E. coli cultures (20 mL) were harvested
hourly during the growth curve by centrifugation at 6000g for 30 min. Pellets were washed twice with distilled water and transferred
onto cellulose acetate membranes (0.45 μm, Whatman) and immediately
dried in a hot air oven for ≥4 h at 90 °C. The mass of
each membrane was measured with an analytical balance before and after
adding the cells, and these data were used to calculate the dry cell
weight per volume.
8
Dot Blot Analysis of Polyglutamine Proteins
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Neural progenitor sphere pellets were lysed by incubation in 20–40 μl SDP buffer for 30 min on ice with vortexing every 5 min. Cellulose acetate membrane (Whatman, 10404180) was equilibrated in 0.1% SDS for at least 10 min. After dot blotter (BioRad, 170-6545) was assembled, 30 μg sample was diluted in 200 μL 2% SDS and boiled for 5 min. Samples were added to each well and vacuum was used to filter the samples through the membrane. After washing with 0.1% SDS, the membrane was blocked for 1 h in 5% milk before immunoblotting using a mouse anti-polyQ antibody (Sigma 3B5H10, P1874; 1:1,000), as above. Anti-mouse secondary antibodies conjugated to peroxidase (Jackson Labs, 715-005-150; 1:10,000) was applied in block for 1 h at room temperature, followed by exposure to chemiluminescence kit (Super Signal West Femto Maximum Sensitivity Substrate, Thermo Scientific, 34095).
9
Detecting Insoluble Protein Aggregates
10
Polysaccharide Molecular Characterization
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Polysaccharides were dissolved in distilled water (2 mg/mL) and heated for 30 s in a microwave bomb prior to molecular measurement (#4872; Parr Instrument Co., Moline, IL, USA). The samples were immediately filtered through a cellulose acetate membrane (3.0 μm pore size; Whatman International). A high performance size exclusion chromatography column (TSK G5000 PW, 7.5 × 600 mm; Toso Biosep, Montgomeryville, PA, USA) linked to a UV detector (Waters, 2487), multi-angle laser light scattering (HELEOS; Wyatt Technology Corp, Santa Barbara, CA, USA) and refractive index detection (Waters, 2414) system (HPSEC-UV-MALLS-RI) were used to analyze the molecular characteristics. The mobile phase was composed of an aqueous solution of 0.15 M NaNO3 and 0.02% NaN3 with the flow rate of 0.4 mL/min. Bovine serum albumin (BSA) was employed to determine the volume delays among the UV, MALLS and RI detectors [23 ].
ASTRA 5.3 software (Wyatt Technology Corp.) was used to calculate the weight average molecular weight (Mw), radius of gyration (Rg) and polydispersity. The specific volume of gyration (SVg) was calculated based on the following equation [24 ]:
in which N is Avogadro’s number (6.02 × 1023/mol) and the units for SVg, Mw and Rg were cm3/g, g/mol and nm respectively.
ASTRA 5.3 software (Wyatt Technology Corp.) was used to calculate the weight average molecular weight (Mw), radius of gyration (Rg) and polydispersity. The specific volume of gyration (SVg) was calculated based on the following equation [24 ]:
in which N is Avogadro’s number (6.02 × 1023/mol) and the units for SVg, Mw and Rg were cm3/g, g/mol and nm respectively.
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