Proximal colons from C57BL/6 mice (Elevage Janvier) were washed with bleach and homogenized in 2 ml of sterile phosphate-buffered saline (PBS) using the Precellys system with 2.8-mm ceramic beads. This mixture was then added to 30 ml of a minimum medium (0.04 M KH2PO4-Na2HPO4 [pH 6], 20 mM KNO3, 0.8 mM MgSO4 ⋅ 7H2O) with 14 mM sodium acetate ⋅ 3H2O as the carbon source (61 (link)). The cultures were incubated at 30°C for 48 h with shaking at 300 rpm/min in a Multitron incubation shaker (Infors). The cultures were then isolated on agar plates (GTCS, MacConkey, Herellea, and Chromagar). Selected colonies were reisolated on Chromagar to ensure that a pure colony was obtained. The colonies were identified using the Biolog system (GEN III microplate for both Gram-negative and Gram-positive bacteria; Biolog, Inc., Hayward, CA, USA). The identification of Acinetobacter, Delftia, and Stenotrophomonas was confirmed by Sanger sequencing of 16S rRNA, rpoB, and gyrB genes after genomic DNA extraction by the Wizard genomic DNA purification kit following the manufacturer’s instructions (Promega). The primers used are listed in Table S1 in the supplemental material.
Multitron incubation shaker
Manufactured by Infors
Sourced in Switzerland
The Multitron incubation shaker is a laboratory equipment designed for temperature-controlled incubation and shaking of samples. It provides a stable and uniform environment for cell culture, microbial fermentation, and other applications requiring controlled temperature and agitation.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Huvecs, by Lonza (2 mentions)
Freestyle expression medium, by Thermo Fisher Scientific (2 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Antibiotic antimycotic, by Corning (1 mentions)
Hct116 cell, by American Type Culture Collection (1 mentions)
Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions)
Cfpac 1, by American Type Culture Collection (1 mentions)
U87 cells, by Korean Cell Line Bank (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Egm 2, by Lonza (1 mentions)
Mccoy s 5a medium, by Corning (1 mentions)
Eagle s minimum essential medium, by Thermo Fisher Scientific (1 mentions)
Zr 75 1, by American Type Culture Collection (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Microtitre plate reader, by Molecular Devices (1 mentions)
6 protocols using multitron incubation shaker
1
Isolation and Identification of Bacterial Isolates
2
Cultivation of Lactobacillus Strains
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Seed cultures of the different strains were prepared by picking a colony from an agar plate and putting it into a test tube with 5 mL MRS medium [31 ]. Lactobacilli were incubated at 30 °C (L. lactis at 37 °C) and stirred at 110 rpm until the exponential phase was reached. Standard cultivations were in general performed with MRS medium at initial pH 6, 30 °C and 110 rpm (Multitron incubation shaker, Infors HT, Switzerland). Cultivations for the determination of the pH tolerance were performed at initial pH values of 4 and 6.
3
Culturing Diverse Cell Lines for Research
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All of the cells were maintained at 37 °C with 5% CO2 unless otherwise noted. Human umbilical vein endothelial cells (HUVECs; Lonza, Allendale, NJ, USA) were cultured in endothelial growth medium‐2 (EGM‐2; Lonza). SNU182 cells (Korean Cell Bank, Seoul, Korea) were cultured in RPMI 1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). CFPAC‐1 (ATCC, Manassas, VA, USA) and U87 cells (Korea Cell Line Bank, Seoul, Korea) were cultured in Iscove's modified Dulbecco's medium (Gibco) with the same supplements. HCT116 cells (ATCC) were cultured in McCoy's 5a medium (Corning, Steuben County, NY, USA) with 10% FBS (Corning) and 1× antibiotic/antimycotic (Corning). Bevacizumab‐adapted HCT116 cells (HCT116/Beva) (MD Anderson Cancer Center, Houston, TX, USA) were cultured in McCoy's 5a medium with 10% FBS, 1× antibiotic/antimycotic, and 250 μg·mL−1 bevacizumab (Genentech/Roche, South San Francisco, CA, USA). HEK293F cells were cultured in Freestyle™ expression medium (Gibco) in a humidified Multitron incubation shaker (Infors HT) with 8% CO2.
4
Cell Culture Protocols for Cancer Research
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Cancer cell lines were maintained at 37 °C with 5% CO2. ZR-75-1 was obtained from American Type Culture Collection (Manassas, VA, USA) and T47D, BT-20, MCF-7, HCT116, and MDA-MB-231 were obtained from KCLB (Korean Cell Bank, Seoul, Korea). ZR-75-1 and T47D cell lines were cultured in RPMI 1640 (Hyclone, Logan, UT, USA) medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L l -glutamine. All the above cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). BT-20 (Korean Cell Bank, Seoul, Korea) cells were cultured in Eagle’s minimum essential medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) as guided by ATCC. HEK293F cells were cultured in FreestyleTM expression medium (Gibco) using Multitron incubation shaker (Infors HT, Bottmingen, Switzerland) with 8% CO2, 37 °C.
5
HUVEC Transfection for VCAM-1 Silencing
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Human umbilical vein endothelial cells (HUVECs; Lonza, Basel, Switzerland) were maintained in endothelial growth medium (EGM; Lonza) at 37 °C in a humidified incubator with 5% CO2 (Panasonic Healthcare Company, Tokyo, Japan). Human embryonic kidney 293F (HEK293F) cells were maintained in Freestyle expression medium (Invitrogen/Life Technologies, Carlsbad, CA, USA) supplemented with 1% (v/v) penicillin/streptomycin in a humidified Multitron incubation shaker (Infors HT, Basel, Switzerland) at 37 °C in 8% CO2. HUVECs were grown to 50–80% confluence and transiently transfected with ON-TARGETplus SMARTpool siRNA targeting VCAM-1 (Thermo Fisher Scientific, Waltham, MA, USA) using Lipofectamine 2000 transfection reagent (Invitrogen), according to the manufacturer's instructions.
6
β-Galactosidase Assay Protocol
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All β-galactosidase assays were performed in our standard two-hybrid reporter strain, FW102 OL2-62. FW102 OL2-62 cells were co-transformed with plasmids encoding the relevant α and λCI fusions. Cultures inoculated with transformants were grown in 1 mL LB (KanCmCarb) in deep-well 96-well plates at 37°C, 900 rpm, 90% humidity in a Multitron incubation shaker (Infors HT) overnight. Overnight cultures were back diluted 1:100 or 1:40 in LB (KanCmCarb) supplemented with the appropriate concentration of IPTG in sterile microtitre plates (total volume of 200 μL); subcultures were grown, shaking at 37°C until they reached mid-log phase (OD600 0.4–0.8). A 100 μL aliquot of subculture was lysed by addition of 10 μL PopCulture reagent (Novagen) supplemented with rlysozyme (400 mU/μL). LacZ levels were determined by β-galactosidase assay performed in microtitre plates with a microtitre plate reader (Molecular Devices), as described in [83 (link)]. All assays were done in triplicate and were repeated independently at least twice. All Miller Unit values shown are from a single representative experiment and represent averages of triplicate measurements. Fold-change values were calculated by normalizing to the highest relevant empty vector control.
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