The virucidal activity of disinfectants was assessed by adding 200 µl virus/soil mixture to 800 µl of the diluted disinfectants mentioned above achieving 70 and 40% final concentrations for ethanol, 0.5 and 0.05% for NaOCl, and 0.2 and 0.02% for PAA, and incubating for 30 s. Subsequently, 50 µl of the virucidal test was inoculated directly into 25 cm2 flasks to determine residual infectivity after treatment. Another 50 µl were added to 450 µl of cell culture media (to dilute ethanol to a non-working concentration) or cell culture media supplemented with neutralizer (1% sodium thiosulfate to neutralize NaOCl) or cell culture media supplemented with both neutralizer (0.25% sodium thiosulfate) and additional 25 mM of HEPES (to neutralize and buffer PAA; Gibco, Fischer Scientific, Reinach, Switzerland) and diluted tenfold in a deep well plate (Eppendorf, Schönenbuch, Switzerland). 150 µl of each dilution was then inoculated on Vero E6 cells (median passage number 42) in a 24-well plate (Techno Plastic Products, TPP, Trasadingen, Switzerland) and incubated at RT for 1 h. After incubation, 1 ml of overlay media, 2% MEM + 1% methylcellulose 90 H G 4000 cP (Sigma Aldrich, Buchs, Switzerland), was added on top and cells incubated at 37 °C without CO2 for 7 days.
24 well plate
Manufactured by Techno Plastic Products
Sourced in Switzerland
24-well plates are a type of laboratory equipment used for cell culture and various experimental procedures. These plates consist of a rectangular array of 24 individual wells, providing a standardized format for conducting multiple experiments or observations simultaneously. The wells are typically designed to hold a small volume of liquid, such as cell culture media or reagents, allowing researchers to work with small sample sizes efficiently.
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Lab products found in correlation
Hepes, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Deep well plate, by Eppendorf (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Dmem glutamax medium, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Round glass coverslip, by Thermo Fisher Scientific (1 mentions)
Gentamycin, by Thermo Fisher Scientific (1 mentions)
Mem neaa 100x, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by BioConcept (1 mentions)
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Olympus (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
48 well plate, by Greiner (1 mentions)
Incucyte device, by Sartorius (1 mentions)
Triton x 100, by Carl Roth (1 mentions)
Cytotoxicity detection kit, by Roche (1 mentions)
Crystal violet, by Merck Group (1 mentions)
Dispase 2, by Merck Group (1 mentions)
18 protocols using 24 well plate
1
Virucidal Efficacy of Disinfectants
2
Genetic Transformation of C. merolae
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The transformation of C. merolae was carried out by the PEG-mediated method [46 (link)], as described previously [2 , 30 (link)], with minor modifications. To generate the BSD-MITmS strain, 4 μg of BSD-MITmS PCR product was introduced into C. merolae WT. To generate the BSD-MITmS/CAT-mV strain, 4 μg each of BSD-MITmS and CAT-mV PCR products were mixed (8 μg in total) and introduced into C. merolae WT.
The transformed cells were cultured for 2 days in 8 mL of MA2 liquid medium in one well of a 6-well plate (VIOLAMO) in a CO2 (3%) incubator at 42 °C in the light (40 μE) for recovery and selected on an MA2 gellan gum plate supplemented with 1, 1.25, and 1.5 mg/mL BS (the preparation procedure is described below) or 2 mL MA2 liquid medium supplemented with both 1 mg/mL BS and 0.2 mg/mL CP (the concentration was defined previously [30 (link)];) in one well of a 24-well plate (TPP Techno Plastic Products). Colony PCR analysis was carried out to verify targeted insertions of constructs into the intergenic regions between CMD184C and CMD185C loci and upstream of the CMK046C locus using the primer sets #11/12 and #13/14, respectively. The BSD marker integration into the genome in transformants was verified by PCR using the primer set #15/16.
The transformed cells were cultured for 2 days in 8 mL of MA2 liquid medium in one well of a 6-well plate (VIOLAMO) in a CO2 (3%) incubator at 42 °C in the light (40 μE) for recovery and selected on an MA2 gellan gum plate supplemented with 1, 1.25, and 1.5 mg/mL BS (the preparation procedure is described below) or 2 mL MA2 liquid medium supplemented with both 1 mg/mL BS and 0.2 mg/mL CP (the concentration was defined previously [30 (link)];) in one well of a 24-well plate (TPP Techno Plastic Products). Colony PCR analysis was carried out to verify targeted insertions of constructs into the intergenic regions between CMD184C and CMD185C loci and upstream of the CMK046C locus using the primer sets #11/12 and #13/14, respectively. The BSD marker integration into the genome in transformants was verified by PCR using the primer set #15/16.
3
Oxidative Stress Tolerance in Worms
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Synchronised worms were cultured on OP or BL plates (0•5 or 5•0 mg/ml) for 96 h and then transferred to 500 µl of 0•3 % H 2 O 2 (Sigma-Aldrich Japan) in a 24-well plate (Techno Plastic Products AG). The time of transfer was designated as the 0 h. The worm survival rate was determined every hour starting from 2 h after the transfer. The survival rate at 0 h was set as 100 %, and 24 worms were assessed per group.
Tolerance to oxidative stress in mgDf50 and tm4211 worms was also evaluated using the method described above. The worms were cultured on OP or BL plates (5•0 mg/ml) and 24 worms were assessed per group.
Tolerance to oxidative stress in mgDf50 and tm4211 worms was also evaluated using the method described above. The worms were cultured on OP or BL plates (5•0 mg/ml) and 24 worms were assessed per group.
4
3D Microtissue Spheroid Formation and Evaluation
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To build three-dimensional microtissue spheroids of GF and PDLF, we procured flexible 3D Petri Dish® (Microtissues Inc., Providence, RI, USA) with 35 spheroidal recesses and poured 330 μl molten agarose (2% agarose powder in 0.9% NaCl) into the molds, under aseptic conditions. After 2 min of gelling, the molds were inverted into alpha-MEM growth medium for conditioning up to 5 min. Molds were carefully transferred into the 24 well plates (TPP Techno Plastic Products, Trasadingen, Switzerland) using sterile forceps, and each mold received 75 μl of cell suspension of GF and PDLF with a cell count of 73 × 105 cells/ml. L929 cells do not form spheroids; hence, they were excluded from this step. Cell settling time of 15 min was followed by the addition of fresh α-MEM 1 ml/well, outside the mold. The plate was incubated for 24 h at 37 °C, and microscopic evaluation confirmed the formation of spheroids over 24 h. Spheroids were cultured directly on LAY-FOMM 40 and LAY-FOMM 60 for the next 24 h. The set-up was then subjected to resazurin-based toxicity assay and MTT and Live-dead staining.
5
Visualizing MERS-CoV Spike Protein Expression
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A total of 1.25 × 105 Huh-7 cells were first seeded on coverslips in 24-well plates (TPP Techno Plastic Products) for 24 h. Plasmids encoded with the spike of EMC/2012, BF785, or an empty vector were transfected in the cells using TransIT-LT1 transfection reagent (Mirus) (25 (link)). At 20 h posttransfection, the cells were fixed with 4% PFA, permeabilized with 0.3% Triton X-100, and immunolabeled using primary polyclonal antibody against MERS-CoV spike protein (SinoBiological) and secondary Alexa Fluor 488-conjugated goat anti-rabbit antibody (Thermo Fisher). Nuclei were stained using DAPI. Images were acquired by a Zeiss LSM710 inverted confocal microscope with a 20× objective. Cell scoring function in Metamorph software was used to quantify syncytia formation by calculating nuclei/syncytium.
6
SH-SY5Y Cell Differentiation and PCL-NP Exposure
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SH-SY5Y cells were obtained from ATCC (Manassas, VA, USA), and culturing as well as differentiation was done with some adaptations as previously described [5 (link)]. Briefly, at day in vitro (DIV) 0, SH-SY5Y cells were seeded at a density of 1 × 107 cells per T75 for Western samples, 8 × 104 cells per well in 24-well plates (Techno Plastic Products AG (TPP) Trasadingen, Switzerland) and maintained at non-differentiated state for 24 h in Dulbecco’s Modified Eagle Medium (DMEM) GlutaMAX™ medium (Life Technologies, UK) sodium pyruvate [1 mM], l -glutamine [2 mM], penicillin/streptomycin ([1 unit/ml], Life Technologies, UK) at 37 °C in an atmosphere of 5% CO2.
For the first 3 days of differentiation, cells were exposed to the same medium with a reduced FBS concentration (5%) and supplemented with retinoic acid [10 μM] (RA, Sigma, St Louis, USA). For the last 3 days, SH-SY5Y cells were grown in DMEM with only 1% FBS with RA [10 μM]. PCL-NP exposure was performed before differentiation (NP DIFF) or during differentiation (DIFF NP DIFF) on DIV1 or DIV4, respectively. The cells were exposed to PCL-NPs in the same medium supplemented with 1% FBS for 24 h.
For the first 3 days of differentiation, cells were exposed to the same medium with a reduced FBS concentration (5%) and supplemented with retinoic acid [10 μM] (RA, Sigma, St Louis, USA). For the last 3 days, SH-SY5Y cells were grown in DMEM with only 1% FBS with RA [10 μM]. PCL-NP exposure was performed before differentiation (NP DIFF) or during differentiation (DIFF NP DIFF) on DIV1 or DIV4, respectively. The cells were exposed to PCL-NPs in the same medium supplemented with 1% FBS for 24 h.
7
Adaptation of L. monocytogenes to Quaternary Ammonium Compounds
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Planktonic cells of L. monocytogenes strains were adapted to fixed or progressively increasing sublethal concentrations of QAC in 24-well plates (Techno Plastic Products, AG, Switzerland) using two broth models and one water model: (1) fixed concentration of QAC in TSBYE (QAC-P1); (2) gradually increasing concentration of QAC in TSBYE (QAC-P2); and (3) gradually increasing concentration of QAC in distilled water (QAC-P3).
8
Culturing Vero and HeLa Cells for Infection Studies
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Vero 76 cells (African green monkey kidney cells, CRL 1587 American Type Culture Collection (ATCC), Manassas, VA, USA) and HeLa cells (Homo sapiens cervix adenocarcinoma, CCL-2 ATCC) were cultured at 37°C with 5% CO2 in growth culture medium for cell propagation. Vero growth medium consisted of Minimal Essential Medium (MEM) with Earle's salts, 25 mM HEPES, without L-Glutamine (GIBCO, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, BioConcept, Allschwil, Switzerland), 4 mM GlutaMAX-I (200 mM, GIBCO) and 0.2 mg/ml gentamycin (50 mg/ml, GIBCO). HeLa cell culture media were further supplemented with 1% MEM Non-Essential Amino Acids (MEM NEAA, 100x, GIBCO). Medium used for cell propagation intended for infection experiments was without gentamycin [13] (link). Cells were seeded on round glass coverslips (13 mm diameter, Sterilin Limited (Thermo Fisher Scientific), Cambridge, UK) in 24-well plates (Techno Plastic Products AG (TPP), Trasadingen, Switzerland) at a density of 3×105/well in 1 mL medium for infection experiments. Infection experiments were performed when cells reached at least 90% confluency.
9
Quantifying IONP Uptake in Chondrosarcoma
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Uptake and retention of IONP in SW1353 was assessed using a previous protocol20 (link). Chondrosarcoma cells were seeded in 24 well plates (TPP Techno Plastic Products AG, Trasadingen, Switzerland) at a concentration of 105 cells/well and treated as described above. Afterwards, cells were washed three times with PBS, detached and centrifuged at 1700 rpm for 5 min, to eliminate the nanoparticles that were not interacting directly with the cells. The supernatant was replaced with nanoparticle free fresh medium and the cells were seeded on glass coverslips at a density of 5 × 104 cells/slide and incubated for an additional 24 h. After this time, the coverslips were washed with PBS and fixed with 4% polyformaldehyde for 10 min. Afterwards, the samples were incubated for 10 min with Hoechst (Invitrogen, Thermo Fisher Scientific, Waltham, USA) at a concentration of 1 μg/mL. Following staining, coverslips were washed with PBS and mounted on a slide with glycerol. Imaging was done using the epifluorescence microscope Olympus BX-51 (Olympus, Germany) taking advantage of DOX autofluorescence, while Hoechst counterstaining of the nuclei was applied.
10
Photodynamic Therapy with Hypericin
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HT-29 cells were adjusted to a density of 5 × 105 cells/mL and 1 mL cell suspension was seeded into 24-well plates (TPP Techno Plastic Products AG, Trasadingen, Schweiz) and incubated for adherence overnight. Jurkat cells were adjusted to a density of 2.5 × 105 cells/mL and 1 mL was seeded into 48-well plates (Greiner bio-one, Frickenhausen, Germany). Then, cells were pre-incubated with free hypericin for 16 h in the dark (plates were wrapped with aluminum foil) to enable uptake by the cells. Methanol-treated cells and phosphate buffered saline (PBS)-treated cells served as controls. Then, cells were illuminated with a slimlite light-emitting diode (LED) white light source (40 W/m2, Kaiser, Buchen, Germany) for various time intervals to start the phototoxic reaction of hypericin.
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