G. duodenalis isolates used in this work are listed in Table 1 . Trophozoites were axenically grown at 37 °C in 10 mL screw-cap tubes (NuncTM, Thermo Fisher Scientific, Waltham, MA, USA) filled with TYI-S33 medium, supplemented with 10% adult bovine serum (Euroclone S.p.A., Milan, Italy) and bovine bile (Sigma-Aldrich, Merck Life Science S.r.l., Milan, Italy) [34 (link)] and sub-cultured into fresh medium when confluence was reached. Where appropriate, parasites were grown in a 50 or 500 mL screw-cap culture flask (NuncTM, Thermo Fisher Scientific, Waltham, MA, USA).
Nunc
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark, United Kingdom
Nunc is a line of high-quality lab equipment manufactured by Thermo Fisher Scientific. The products are designed for various applications in scientific research and laboratory settings. Nunc offers a range of products, including cell culture plates, dishes, flasks, and tubes, that are engineered to provide consistent and reliable performance.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (5 mentions)
Dispase 2, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Resveratrol, by Merck Group (3 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Indomethacin, by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
α mem, by Thermo Fisher Scientific (3 mentions)
Collagenase type 1, by Worthington (3 mentions)
T 75 cm 2 ask, by Thermo Fisher Scientific (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Ascorbate 2 phosphate, by Merck Group (2 mentions)
Bx51 microscope, by Olympus (1 mentions)
Tcs sp5, by Leica (1 mentions)
Foetal bovine serum (fbs), by Bovogen (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
46 protocols using nunc
1
Axenic culture of Giardia duodenalis
2
Evaluating Antibody-Mediated Pneumococcal Killing
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The serum bactericidal assay (SBA) as a functional antibody test was performed to evaluate whether the specific antibodies created in each group have an in vitro bactericidal activity using the complement system or not. In addition, the titer of required antibodies to kill bacteria following complement activation was measured (43 (link)). For this purpose, the 96-well polystyrene round bottom microwell plates (Thermo scientific NuncTM, USA) were loaded for 30 min at 37°C with 12.5μl of prepared pneumococcal suspension (S. pneumoniae ATCC 49619) at 106 CFU/ml (based on the standard of 0.5 McFarland) and 12.5μl of dilutions of inactivated serum samples at 56°C for 30 min (1:2 to 1:64). Then, infant rabbit serum (4%) as a source of complement was added to each well. At two intervals (0 and 2 h), the content of each reaction well was serially diluted, and 10 μl from each well was inoculated into the blood agar plate. After 18-24 hours of incubation at 37°C in 5% CO2, the colony-forming unit of bacteria was measured. The titer of bactericidal antibodies is usually considered as the dilution of serum, which killed 50% of pneumococci compared to the control. The negative control was a well that contained bacteria and rabbit complement (23 (link), 36 (link), 40 (link), 44 (link), 45 (link)).
3
Tumor Cell Migration Assessed by Wound Assay
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Tumor cell migration was assessed by means of the wound assay model [31 (link)]. The cell lines were grown at 2.5 × 105 in standard conditions (37 °C, 95% humidity, 5% CO2) in 4 cm-diameter culture dishes (NuncTM, ThermoFisher, Rochester, NY, USA). The next day, the cell monolayer was scratched with the pipette tip (P300), the medium and dislodged cells were aspirated, and the plates were rinsed twice with PBS. Next, fresh culture medium was applied and the number of cells that had migrated into the wound area after 24 h was estimated in the control and drug-treated cultures. The plates were stained with the May–Grünwald–Giemsa method. The observation was performed with the use of a BX51 microscope (Olympus, UK), and the distances between the scratch fronts were estimated using the CellSans program. The results were presented as the migratory potential expressed as the percent of cells within the wound.
4
Cellular Uptake of InP/ZnS Quantum Dots
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The day before imaging, cells were planted onto 35 mm confocal dishes (Thermo ScientificTM NuncTM, United States) to give 30–50% density. Cells were left untreated or co-incubated with 2 μg/mL InP/ZnS QDs. After 4–6 h of incubation, the culture medium was removed and cells were washed with phosphate-buffered saline (PBS) twice, fixed with 4% paraformaldehyde for 15 min. Then the paraformaldehyde solution was abandoned and the cell nucleuses were stained with DAPI for 5 min. The confocal images were obtained using a laser scanning confocal microscope (LSCM, TCS SP5, Leica, DEU).
5
Culturing Human Cancer Cell Lines
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Cell culture plates for adherent cells were purchased from NuncTM (ThermoFisher Scientific, Waltham, MA, USA). The human breast tumour cell lines MDA-MB 231 (ER−) and MCF-7 (ER+), and human prostate tumour cell lines PC-3 (AR−/PSA−) and LnCap (AR+) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in RPMI-1640 medium (#11875-093, Gibco, ThermoFisher Scientific) supplemented with 10% foetal bovine serum (#SFBS, Bovogen, Victoria, Australia) and 100 U/mL PenStrep (#15070063, Life Technologies, Carlsbad, CA, USA). All cells were maintained at 37 °C in a humid incubator with 5% CO2.
6
Chondrocyte Differentiation Monitoring Protocol
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The suspensions of CTC, SOX9-TC, IGF-I-TC, and NTC
were adjusted to densities of 2×104 cells/mL in complete medium, and 500
µL aliquots were seeded into each compartment of a four-well microchamber slide
(NuncTM; Thermo Fisher Scientific). One microchamber slide was prepared for each
transfection condition, and the microchamber slides were immediately incubated at
37°C in a 5% CO2 atmosphere for 3, 6, or 9 days. Next, the culture medium
was removed and the cells were fixed with methanol-acetone (1:1 v/v) for 20 min at
-20°C. The NTC, IGF-I-TC, SOX9-TC, and CTC on the
3rd, 6th, and 9th PT days were incubated with monoclonal antibodies against both
Col-I (ab23446; Abcam, Inc., USA) and Col-II (ab34712; Abcam, Inc.), and positive
staining was detected using mouse- and rabbit-specific HRP/DAB detection IHC Kit
(Abcam, Inc.), according to the manufacturer's instructions.
were adjusted to densities of 2×104 cells/mL in complete medium, and 500
µL aliquots were seeded into each compartment of a four-well microchamber slide
(NuncTM; Thermo Fisher Scientific). One microchamber slide was prepared for each
transfection condition, and the microchamber slides were immediately incubated at
37°C in a 5% CO2 atmosphere for 3, 6, or 9 days. Next, the culture medium
was removed and the cells were fixed with methanol-acetone (1:1 v/v) for 20 min at
-20°C. The NTC, IGF-I-TC, SOX9-TC, and CTC on the
3rd, 6th, and 9th PT days were incubated with monoclonal antibodies against both
Col-I (ab23446; Abcam, Inc., USA) and Col-II (ab34712; Abcam, Inc.), and positive
staining was detected using mouse- and rabbit-specific HRP/DAB detection IHC Kit
(Abcam, Inc.), according to the manufacturer's instructions.
7
Hemolytic Activity Assay of Compounds
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Fresh human blood was collected from a healthy donor (age 30, Male). 1 mL blood was mixed with 9mL PBS and centrifuged at 1,000 rpm for 5 min. Red blood cell pellets were collected and subsequently washed with PBS three times and diluted to a final concentration of 5% v/v. The COE was dissolved in PBS and two folds serial diluted in a 96-well microplate (NuncTM, ThermoScientific). 50 μL red blood cell stock was mixed with 50 μL COE solution in each well and incubated for 1 h at 37°C under shaking. The microplate was centrifuged at 1,000 rpm for 10 min. 80 μL aliquots of the supernatant were then transferred to a new 96-well microplate and diluted with another 80 μL of PBS. Hemolytic activity was calculated by measuring absorbance at 540 nm using a 96-well plate spectrophotometer (Benchmark Plus, BIO-RAD). Triton X-100 (0.1% in PBS) which is able to lyse red blood cells completely was used as positive control, while PBS was used as negative control. The hemolysis percentage was calculated using the following formula:
where Oc is the absorbance of COE-treated sample, Ob is the absorbance of negative control and Ot is the absorbance of positive control. The results were repeated in four replicates.
where Oc is the absorbance of COE-treated sample, Ob is the absorbance of negative control and Ot is the absorbance of positive control. The results were repeated in four replicates.
8
Immunofluorescence Assay in MDA-MB-468 Cells
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MDA-MB-468 cells were plated in 96-well Terasaki plates (NuncTM; Thermo Fisher Scientific, Roskilde, Denmark), and immunofluorescence was performed as previously described [38 (link)]. A volume of 12 μl per Terasaki well was used.
9
Quantifying Cell Proliferation Dynamics
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A total of 1000 COLO320DM and HCT-15 cells were plated in six parallels per treatment in a 96-well plate (NuncTM,Thermo Fischer Scientific, Boston, MA, USA). The following day, culture media was replaced with vehicle or inhibitor containing media. Cell confluence was quantified with the IncuCyte live-cell analysis system (Essen BioScience, Ann Arbor, MI, USA). Images were captured every second hour to monitor proliferation and results were retrieved through corresponding IncuCyte 2011A software (Essen BioScience), and exported to Excel for further analysis and graph design.
10
Quantification of HCV Pseudoparticle Transduction
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Confluent Huh7 cells monolayers grown in 100 × 10 mm dish (nuncTM, Thermo Fisher Scientific, China) were transduced with 90 µl HCVpp supplemented with DMEM (910 µl DMEM and 90 µl HCVpp) without FBS and antibiotics on ice for 1 h. Infected dish was incubated for 20 min in 5% CO2 at 37 °C followed by wash with PBS twice. Post internalization cells were scraped and resuspended in 1 ml PBS on ice, followed by a centrifugation at 500xg for 2 min. Total RNA was isolated using Trizol reagent (Sisco research laboratories, India) with 400 µg Ribonucleoside vanadyl complexes (VRC) from NEB, USA. cDNA was synthesized with 1 μg of RNA, using iScript™ Reverse Transcription supermix for qRT-PCR kit (BIO-RAD Laboratories, USA) following manufacturer’s instructions at PCR conditions (priming at 25 °C for 5 min, reverse transcription at 46 °C × 20 min, inactivation at 95 °C × 1 min at RT (25 °C)). 60 ng of cDNA was amplified by PCR using (20 pmol/ reaction) forward and reverse primer for KGFP (forward 5′TCGACAGTCAGCCGCATCT3′ and reverse primer 5′CCGTTGACTCCGACCTTCA3′) respectively using 1 µl Taq polymerase (NEB, USA). The PCR amplification was performed as per the following cycle: 95 °C × 5 min, 30 cycles of 95 °C × 30 s, 47 °C × 45 s and 72 °C × 1 min followed by a final extension at 72 °C for 5 min. The PCR amplified product was checked in 1.5% agarose gel electrophoresis.
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