Serum samples were diluted 10-fold in Dulbecco's modified Eagle's medium (DMEM) (ATCC) with 1% FBS (Sigma) and added to a Vero E6 (ATCC) monolayer in a 6-well plate (Costar; Corning). Plates were incubated for 1 h at 37°C and rocked every 15 min. Following incubation, a 1:1 mixture of 1% agarose (Lonza) in 2× medium 199 (Invitrogen) was added on top of the monolayer. After 4 days of incubation at 37°C, a 1:1 mixture of 1% agarose and 2× medium 199 containing a 1:50 dilution of neutral red (Fisher) was added on top of the previous agarose layer. The following day, plaques were counted.
Agarose
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Canada, China, France, Sweden, Spain
Agarose is a polysaccharide derived from red seaweed. It is a versatile laboratory material used in various applications, such as gel electrophoresis and chromatography. Agarose forms a porous gel matrix that allows for the separation and purification of biomolecules, such as DNA, RNA, and proteins, based on their size and charge.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (17 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (13 mentions)
Agarose, by Merck Group (10 mentions)
Ethidium bromide, by Thermo Fisher Scientific (10 mentions)
Bovine serum albumin (bsa), by Merck Group (9 mentions)
Crystal violet, by Merck Group (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Sodium hydroxide (naoh), by Merck Group (7 mentions)
Deae dextran, by Merck Group (7 mentions)
Glycerol, by Merck Group (7 mentions)
Dntps, by Thermo Fisher Scientific (7 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (6 mentions)
Triton x 100, by Merck Group (6 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Sucrose, by Merck Group (6 mentions)
Mgcl2, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
403 protocols using agarose
1
Virus Plaque Assay Protocol
2
Biomimetic Hydrogel Scaffold Fabrication
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Agarose (Fisher Scientific, Loughborough, UK) was mixed with water to a final concentration of 0.75% (w/v) with different concentrations of hydroxyapatite (HA) nanoparticles (Sigma-Aldrich, Gillingham, UK), namely 0% (v/v) for the muscle area, 0.2% (v/v) for the tendon area, and 40% (v/v) for the bone area. Type I rat tail collagen (Corning, Gillingham, UK) was prepared following the company’s instruction to a final concentration of 3 mg/mL. Therefore, the required volumes of sterile 10x phosphate buffer saline (PBS), sterile 1 N sodium hydroxide (NaOH) (Fisher Scientific, UK) in distilled water (dH2O), and Agarose/HA solutions were calculated and mixed for each section in individual tubes. To avoid very rapid polymerization of Agarose, the solutions were kept in a water bath at 37 °C. Additionally, collagen was kept at 4 °C and added last to the mixture. After polymerization, bone and tendon triphasic gels were crosslinked with 10% (v/v) oligomeric proanthocyanidins (OPC) in 1x PBS for 60 min at 37 °C and 5% CO2. The resultant hydrogels are listed in Table 1 .
3
Viral Plaque Assay for Influenza Virus
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For viral plaque assay, confluent MDCK cells were propagated in 6-well plates. Confluent monolayers of MDCK cells were prerinsed with PBS and infected with serially diluted cell culture supernatant (800 μL per well) collected from the HBEpC cells exposed to influenza virus. Infected cells were incubated for 45 min at 35 °C. Cells were then washed with PBS and overlayed with 0.6% agarose (Oxoid Ltd., Hampshire, UK) in DMEM/F12 medium, and plates were kept at 35 °C for another 60 h. Following the incubation, cells were treated with 10% formalin and agarose layer was removed. Cells were stained with 1.0% crystal violet and plaque-forming units (PFU) were counted.
4
Fixation and Embedding of Mammospheres
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After 7 to 10 days in culture, mammospheres were gently centrifuged at 200 rpm for 2 minutes at RT. The pellet was then resuspended in formalin and allowed to fix for 1 hour at RT. After fixation, mammospheres were centrifuged, and part of the supernatant was removed. An equal volume of melted 4% agarose (Gibco electrophoresis grade) in distilled water was then added to the formalin-fixed pellet (to a final agarose concentration of 2%). The volume was aspirated in a 1-ml syringe on which the top had been cut off and allowed to cool for 30 minutes at RT before the gel was expelled by using the syringe plunger. The gel was wrapped in lens tissue and paraffin/wax processed after routine tissue-processing procedures. Staining of sectioned mammospheres was done as described for paraffin sections.
5
Total RNA Extraction from Cell Cultures
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After incubation, the cells were collected from the culture dishes and total RNA was extracted by the use of Trizol® reagent in accordance with the manufacturer's instructions. The amount of extracted total RNA was quantified by established optical methods at A260/A280 (Genequant II, Pharmacia Biotech, Freiburg, Germany) and structural integrity checked by agarose-gel electrophoresis [1.5% agarose (Gibco/BRL)].
6
Soft-agar Assay for Cancer Cell Colonies
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Soft-agar colony formation was done as described previously11 (link),76 . Briefly, 2500 cells were resuspended in 0.35% agarose (Invitrogen, USA) mixed with complete growth medium and layered on top of a 1% agarose: growth medium mix. Colonies were allowed to form for 7 weeks (6 weeks for MDA-MB-231 LTC) and the agarose layer was kept hydrated by addition of 200 µL of growth medium (RPMI + 10% FCS) every 5 days. Colonies were stained with 0.005% crystal violet (Fisher science education, USA) solution overnight followed by imaging using an EPSON scanner. Soft-agar colonies formed by MDA-MB-231 LTC cells were difficult to visualize on the EPSON scanner. Therefore, representative images were captured using a ZEN AxioObserver microscope on a ×10 objective.
7
Soft Agar Clonogenic Assay
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1.2 % Agarose (Invitrogen, Carlsbad, CA, USA) and 0.7% Agarose were used, respectively, as upper stratum and underlay. The 104 cells in each group were treated with supplemented with culture medium (10% fetal bovine serum and pre-warmed DMEM medium). Then the above cells were plated in 60mm dishes. Specifically, before the cells were seeded in the plates, an equal volume of 1.2% argorse contained was added to the plates. When the underlay Agarose solidified at room temperature, the above cells was seeded in plates in a mixture of 0.7 % argorse and culture medium. Then each plate was incubated in incubator (37°C, 5 % CO2) for 15 d. The clones (approximately 50 cells in each clone) were identified and counted under the microscope (DM3000, Leica, Solms, Germany).
8
Hydrogel-based Microfluidic Device for Cell Chemotaxis
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A solution of Poly-D-lysine (PDL) (VWR, Radnor, PA, USA) in Milli-Q® water in the concentration of 0.5 mg/mL was injected into the two gel channels of a freshly assembled device, incubated at room temperature for 30 min, and aspirated with vacuum. 1× PBS was injected to the gel channels and aspirated out to remove excess PDL. A solution of agarose (Catalog number: 16500100; Invitrogen, Carlsbad, CA, USA) in the concentration of 0.8% wt/vol was freshly dissolved in water by microwaving for 45 s. Hot agarose solution was injected to the pre-coated hydrogel channels in the same fashion and allowed to solidify at RT for 30 min in a wet chamber to minimize hydrogel dehydration. After the formation of two gel barriers, lipid bilayer formation and/or protein capturing, cell seeding was performed in the center channel for chemotaxis studies (details described in corresponding method sections below).
9
Anchorage-Independent Cell Growth Assay
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The bottom layer was prepared by mixing 1.6% agarose (Invitrogen) with DMEM (1:1; v/v) and adding the mixture into 24-well cell culture plates (500 μL) for 10 min until solidification. Then, the top layer containing MHCC97H, SMMC-7721 or HepG2 cells or respective HCSLCs (500 cells) incubated with or without isovitexin and 0.4% agarose (Invitrogen) in 500 μL of 20% FBS DMEM were placed over the bottom layer. Colonies were counted under an inverted microscope (Olympus CK40; Olympus Corp., Tokyo, Japan) after incubation at 37 °C for 21 days. The colony formation rate (%) was determined by dividing the number of colonies by that of cells seeded, multiplied by 100%.
10
Molecular Detection of P. multocida
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Template nucleic acid was isolated using boiling method as described by Sujatha et al 2018. P. multocida specific PCR (PM-PCR) was done using a set of primers as reported by Townsend et al. (1998) . A 10µl reaction mixture was used as described by Sujatha et al., 2018 with PCR conditions initial denaturation 94°C for 3 min followed by 30 cycles of denaturation 94°C for 30 sec, annealing for 57°C for 40 sec, extension 72°C for 60 sec and final extension 72°C for 10 min. The amplified gene products were subjected to agarose gel electrophoresis using 1.5% agarose (Invitrogen, UK) and then visualised by UV gel documentation system ((Bio-Rad).
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