Approximately 3 mL of sample (previously weighed) taken from the bioreactor was passed through a preweighed 0.2-μm filter (Pall Corporation, USA) with the help of a vacuum filtration pump. Filters were then washed with approximately 40 mL of demineralized water and dried at 80°C for 48 h. Afterward, the filters were weighed on an analytical balance again to calculate the cell dry weight (CDW) concentration of the sample. CDWs were measured in duplicate for every sampling point.
0.2 μm filter
Manufactured by Pall Corporation
Sourced in United States
The 0.2-μm filter is a lab equipment product designed for filtration purposes. It features a pore size of 0.2 micrometers, which allows for the removal of small particles and microorganisms from various liquids or gases. The filter's core function is to provide efficient filtration to support various laboratory applications.
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Lab products found in correlation
Ultrasonic cell grinder, by Ningbo Scientz Biotechnology (1 mentions)
Ebm 2, by Lonza (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Labsolutions software, by Shimadzu (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Pierce chromogenic endotoxin quant kit, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions)
Ebm 2 medium, by Lonza (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Cyquant ldh cytotoxicity assay, by Thermo Fisher Scientific (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
12 protocols using 0.2 μm filter
1
Measuring Microbial Cell Dry Weight
2
Optimizing BoHV-1 Virus Yield in MDBK Cells
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BoHV-1 was passaged 3 times in suspended MDBK cells, quantitated and designated as the seed virus. Suspension-adapted MDBK cells at the densities of 2.0 × 106 cells/mL, 4.0 × 106 cells/mL and 8.0 × 106 cells/mL were infected with the seed virus at the multiplicity of infection (MOI) of 0.1, 0.05 or 0.01. Cells were then harvested with medium at different time points post infection, frozen and thawed 3 times, and the supernatant was stored for virus titration with the protocol described above.
Furthermore, the following methods for virus release were compared for their effects on the recovery of viruses from the MDBK cells. (1) Repeated freeze–thaw cycles (freezing at −80 °C, thawing at room temperature). (2) Ultrasonic treatment. The sonication was performed with the ultrasonic cell grinder (Scientz, Ningbo, China) and the output power was 180 W. The working volume was 0.5 to 1.0 mL, and the infected culture was treated for 1 to 10 min. Additionally, the treated virus suspension was then centrifuged at 3000 rpm for 5 min and the supernatant was filtered by 0.2 μm filter (Pall Corporation, Port Washington, NY, USA) for virus titer determination.
Furthermore, the following methods for virus release were compared for their effects on the recovery of viruses from the MDBK cells. (1) Repeated freeze–thaw cycles (freezing at −80 °C, thawing at room temperature). (2) Ultrasonic treatment. The sonication was performed with the ultrasonic cell grinder (Scientz, Ningbo, China) and the output power was 180 W. The working volume was 0.5 to 1.0 mL, and the infected culture was treated for 1 to 10 min. Additionally, the treated virus suspension was then centrifuged at 3000 rpm for 5 min and the supernatant was filtered by 0.2 μm filter (Pall Corporation, Port Washington, NY, USA) for virus titer determination.
3
Chlorin e6 Preparation and Handling
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High-purity (>98%) lyophilized chlorin e6 trisodium salt (Ce6) (Fotolon; Apocare Pharma GmbH, Bielefeld, Germany) was handled in the dark to restrict ambient light exposure. A stock solution of Ce6 (6 mg per ml) was prepared in double-deionized sterile water (ddH20) and passed through a 0.2-μm filter (Pall Corporation, Ann Arbor, MI). Single-use aliquots were stored at −20°C. Working concentrations (1 μM to 100 μM) were prepared in sterile physiologic saline (SPS; 0.9% NaCl, pH 7.2) immediately prior to the start of each experiment.
4
Preparation of MSC-Conditioned Media
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MSCs were plated at 4 × 104 cells/cm2 in a T25 flask overnight. After washing twice with PBS, cells were cultured with 10 ml EBM2 (Lonza) for another 2 days. Their conditioned media (CMs) were then collected, centrifuged at 400 × g for 10 minutes to remove the cell debris, filtered through a 0.2 μm filter (Pall Corporation, Ann Arbor, MI, USA), and frozen at –80 °C for further studies. MSCs derived from three donors were used.
5
Arbovirus Detection in Mosquitoes
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Pre- and post-feeding blood samples and inoculum at the beginning and end of injection period were inoculated as 10-fold dilutions in the wells of a 96-well microtiter plate seeded with C6/36 cells. Plates were incubated at 28 °C for 7 days before being fixed with 20 % acetone and stored at -20 °C. The bodies and legs + wings from all experiments, and females and males collected in pools from the vertical transmission experiment were homogenized in a QIAGEN TissueLyser II (Qiagen, Hilden, Germany), centrifuged at 14,000 g before being filtered through a 0.2 μm filter (Pall Corporation, Ann Arbor, MI). The body filtrate and filtered saliva expectorates were inoculated as 10-fold dilutions in the wells of a 96-well microtiter plate containing confluent monolayers of C6/36 cells. Legs + wings and mosquito pools were inoculated in quadruplicate onto C6/36 cell monolayers within a 96-well microtiter plate. Plates were incubated, fixed and stored as described above. Presence of virus in fixed plates was determined by fixed cell enzyme immunoassay using specific monoclonal antibodies 3D6 for PCV [4 (link)] and 4G2 for WNV [16 (link)].
6
Quantitative Analysis of MHD Biomarkers
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Nine marker components of MHD (ephedrine HCl, amygdalin, 6-gingerol, glycyrrhizin, liquiritin apioside, liquiritin, cinnamaldehyde, cinnamic acid and coumarin) were analyzed by HPLC (Shimadzu, Corp., Kyoto, Japan). Chromatographic data were analyzed utilizing LabSolutions software (Version 5.54 SP3, Shimadzu). Next, the nine components were subjected to chromatographic separation using a SunFire C18 column. For simultaneous determination, 100 mg of the freeze-dried modified MHD powder dissolved in 20 mL of DW was extracted for 20 min at ambient temperature with the help of an ultra-sonicator (Branson 8510, Danbury, CT) and filtered through a 0.2-μm filter (PALL Life Sciences, MI).6 (link)
7
Cytokine Profiling of Infected Cell Cultures
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Following 4 h of infection, spent media was removed, the wells washed with PBS, and fresh tissue culture media added. At 24, 48, and 72 h post-infection, cell culture supernatants were removed and filtered through a 0.2 μm filter (PALL Life Sciences). Bovine serum albumin (BSA; Sigma) was added at a final concentration of 0.5% (w/v) and the samples were stored at −80 °C. The cytokine levels in supernatants were measured using a Bio-Plex Pro Human Cytokine Multi-Plex Panel (Bio-Rad) in a Bio-Plex 200 System (Bio-Rad), according to the manufacturer's instructions.
8
Trichoderma asperellum Against Phytopathogens
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T. asperellum ACCC30536 was obtained from the Agricultural Culture Collection of China. The five phytopathogens were R. solani, F. oxysporum, S. sclerotiorum, A. alternata and C. chrysosperma, and they were stored at the Laboratory of Forestry Protection, Northeast Forestry University, Harbin, China. T. asperellum was cultured on potato dextrose agar (PDA) slant culture medium at 28 °C for 7 days and stored at 4 °C. A. alternata, S. sclerotiorum, R. solani, C. chrysosperma and F. oxysporum were inoculated in 200 mL of 1/4, 1, 1/2, 1 and 1/2 strength PD medium and cultured in shake flasks for 10 days at 28 °C with shaking at 200 rpm. The medium was filtered using 0.2 μm filters (Pall Corporation, MI, USA) to remove spores, and the filtered fermentation liquid from each pathogen was combined, mixed, and stored in tubes at − 20 °C.
9
LNP Formulation and Characterization for Base Editor mRNA Delivery
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The base editor mRNA and gRNA were coformulated at a weight ratio 1:1 in LNPs. For LNPs containing a combination of two gRNAs, the mRNA:g37:g40 ratio was adjusted to 1:0.5:0.5. The formulations were generated by mixing an aqueous solution of the RNA (pH 4.0) with the four lipid components, a proprietary ionizable lipid, dioleoylphosphatidylethanolamine, cholesterol, and DMG-PEG2000, in ethanol solution. The two solutions were mixed in the microfluidics device from Precision Nanosystems. The LNPs were dialyzed overnight against 1× Tris-buffered saline at 4°C, further concentrated in 100,000 molecular weight cutoff Amicon Ultra centrifugation tubes (Millipore Sigma) and subsequently filtered through 0.2-μm filters (Pall Corporation). Particle size was assessed using the Malvern Panalytical Zetasizer. Endotoxin was measured using the Pierce Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific) following the manufacturer’s protocol.
10
VCAM-1+/- CV-MSCs Promote HUVEC Proliferation
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Pairs of 106 VCAM-1+/−CV-MSCs were incubated in EBM2 medium (Lonza) for 48 hours. Then their conditioned mediums (CMs) were collected, centrifuged at 1800 rpm for 10 minutes to remove cell debris, filtered through 0.2 μm filters (Pall Corporation, Ann Arbor, MI, USA), and frozen at –80 °C. To determine the pro-proliferative effect, VCAM-1+/−CV-MSCCM supplemented with 2 % FBS were used to culture HUVECs for 72 hours. EBM2 supplemented with 2 % FBS, and EGM2-MV (endothelial cells commercial culture medium; Lonza) served as the negative and positive control, respectively. The Cell Counting Kit 8 (Dojindo, Rockville, MD, USA) method was used to measure HUVEC proliferation at 24, 48, and 72 hours. ΔOD450 indicated the final data after subtracting the background. Each sample was performed in quadruplicate.
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