Vero E6 cells grown in 96-well tissue culture plates (Corning) were inoculated with VSVΔG-EBOV and VSVΔG-MARV (MOI = 1.0). After adsorption for 1 h, the inoculum was completely removed. One hundred microliters of growth medium (DMEM supplemented with 10% FCS) were added into each well and then incubated for 33 h at 37°C. Supernatants of the culture medium were collected at 3 h intervals and frozen at -80°C until use. To check the presence of infectious virus particles in the collected supernatants, confluent monolayers of Vero E6 cells on 96-well tissue culture plates (Corning) were inoculated with the supernatant collected at each time point. After incubation for 3 days at 37°C, virus infection was assessed by the presence of CPE. This assay enabled us to predict the time when infected cells shift from non-virus-producing to virus-producing cells (i.e., eclipse phase).
96 well tissue culture plate
Manufactured by Corning
Sourced in United States
The 96-well tissue culture plates are a standard laboratory equipment used for cell culture experiments. These plates provide a uniform and consistent environment for culturing cells in a multi-well format. Each plate contains 96 individual wells, allowing for the simultaneous testing of multiple samples or conditions. The plates are made of tissue culture-treated polystyrene, providing a suitable surface for cell attachment and growth. The 96-well format enables efficient use of limited sample volumes and reagents during cell-based assays and experiments.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Celltiter glo, by Promega (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Heat inactivated fetal bovine serum, by Thermo Fisher Scientific (2 mentions)
Wallac 1420, by PerkinElmer (2 mentions)
Calf serum, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
F12 medium, by Thermo Fisher Scientific (2 mentions)
Prism 5, by GraphPad (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
6 well tissue culture plate, by Corning (2 mentions)
Celltiter blue ctb cell viability assay, by Promega (2 mentions)
Infinite 200 pro, by Tecan (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
144 protocols using 96 well tissue culture plate
1
VSV-based Viral Kinetics Assay
2
Evaluating FliC180-LcrV Fusion Stimulation
3
Cytotoxicity Assay for Ovarian Cancer
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Murine OVCA cell line ID8 and human OVCA cell lines A2780.IP2, A2780.CP20 (platinum resistant), and SKOV3.IP were plated in 96-well tissue culture plates (Corning Costar, NY) at 2000 cells/well in 45μl RPMI + 10% FBS. The plates were incubated at 37 °C in a humidified 5% CO2 atmosphere and treated on day 2 with a single dose of entinostat (LC Laboratories, Woburn, MA), panobinostat (LC Laboratories), azacytidine (TOCRIS, Bristol, UK) or entinostat combined with azacytidine. Testing was done in duplicate assays with 8 replicates each. Cells were collected after either 24 or 72 hours of drug exposure and cytotoxicity was evaluated using the ATPlite luminescence-based assay (PerkinElmer, Waltham, MA) as previously described [33 (link)].
4
Infection of Human Neutrophils with Mycobacterium tuberculosis
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Infection of PMN with H37Rv strain was performed according to Morris et al. [16 ] with minor modifications. Briefly, purified PMN were suspended in RPMI 1640 medium supplemented with 10% FBS. 1 × 105 cells were added to each well of 96-well tissue culture plates (Corning Costar) previously coated with poly-L-lysine (Sigma Aldrich) and incubated for 18 h at 37°C, 5% CO2. Then, cells were gently washed once with warm PBS 1X to remove nonadherent cells. Attached PMN were treated for 1.5 h with the CM and infected with H37Rv at a MOI of 3 for 1 h to allow phagocytosis and uptake. Infected PMN were washed 3 times with warm PBS 1X to eliminate nonphagocytosed bacteria and treated with lysis buffer (SDS 0.1%) for 10 min at room temperature followed by addition of neutralization buffer (BSA 20%). To evaluate the replication of H37Rv within PMN, infected PMN were cultured for further 18 h in RPMI 1640 medium supplemented with 5% FBS and gentamicin (50 μg/ml, Sigma Aldrich) which allow the clearance of the remaining extracellular bacteria. Then, cells were lysed as previously described. In both cases, an aliquot of this suspension was used to determine the CFU by plating serial dilutions on 7H11/OADC (BD Bioscience) agar plates after 21 days growth.
5
MTT Assay for IPEC-J2 Cell Viability
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Cell viability was determined by the previous MTT assay [51 (link)]. IPEC-J2 cells were seeded at 4000 cells/well in 96-well tissue culture plates (COSTAR, Corning, NY, U.S.) in the cytotoxic experiment. After the cells were adhered for 24 h at 37 °C and 5% CO2, the supernatants were discarded. The cells were washed with PBS 3 times, and then 100 μL DMEM/F12 containing the different ingredients without serum and antibiotics in 6 groups was added to the plates according to the above design. The cells were exposed to ZEA (500 and 1000 µg/L) and/or AFB1 (40 and 80 µg/L) for 24 h. At the end of the treatments, each well was added with 10 μL MTT solution (5 mg/mL in PBS), and 4 h later the medium was discarded. Thereafter, 150 μL DMSO was added to each well, and the plates were shaken for 10 min to dissolve the purple formazan crystals. Absorbance (A) was measured at 490 nm (A490) and 630 nm (A630) by using an ELx 800 microplate reader (BIO-TEK Instruments Inc., Winooski, VT, U.S.). Cell counting was conducted using a hemacytometer. The relative viability of the IPEC-J2 cells treated with mycotoxins was expressed as the ratio of optical density (OD), compared to the control group.
6
Cell Proliferation Assay for Human Renal Mesangial Cells
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Human renal mesangial cell were seeded in 96‐well tissue culture plates (Costar) at a density of 1 × 105 cells/mL in 100 µL MsCM. Following starvation in serum‐free MsCM for 24 hours, cells were washed and stimulated with 0.1 ng/mL human IL‐6 for 12, 24 or 36 hours. Controls were incubated with MsCM only. Cell proliferation was assessed using the cell counting kit‐8 (CCK‐8) (Dojindo). Briefly, 10 µL of CCK‐8 was added to each well and incubated at 37°C for 2 hours. Thereafter, the absorbance was measured at 450 nm using a microplate reader (iMark; Bio‐Rad). In the co‐culture system, HRMC were co‐cultured with H/R HK‐2 cells in six‐well plates for 24 hours. Subsequently, the method described above was used to detect HRMC proliferation.
The experiments were performed in triplicate and repeated three times. The % cell proliferation was calculated as follows:
The experiments were performed in triplicate and repeated three times. The % cell proliferation was calculated as follows:
7
Lentiviral Transduction of Murine Macrophages
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RAW264.7, the murine macrophage cell line, was obtained from American Type Culture Collection (ATCC Number: TIB-71). The cells were seeded and cultured in 96-well tissue culture plates (Costar, Cambridge, MA, USA) in DMEM medium with 10 % fetal bovine serum (FBS) (Hyclone). Cells were incubated overnight in a humidified incubator at 37 °C with 5 % CO2 to improve the adherence of cells to the plates. Trypan blue dye exclusion was used for cell quantitation and the viability assays, with viability exceeding 98 % for all trials.
For lentivirus transduction, RAW 264.7 macrophages were infected with either TNF-alpha-RNAi-LV or NC-GFP-LV in serum-free growth medium with 5 mg/ml polybrene at multiplicities of infection (MOI) of 150, which was optimized from the toxicity curve before transduction. After 8 h’ of culturing, the medium was substituted by fresh DMEM complete medium, in order to remove debris and inactive lentiviruses before the cells were continuously incubated for another 72 h. Three days after transfection, the transfection efficiency was examined by fluorescent microscopy.
For lentivirus transduction, RAW 264.7 macrophages were infected with either TNF-alpha-RNAi-LV or NC-GFP-LV in serum-free growth medium with 5 mg/ml polybrene at multiplicities of infection (MOI) of 150, which was optimized from the toxicity curve before transduction. After 8 h’ of culturing, the medium was substituted by fresh DMEM complete medium, in order to remove debris and inactive lentiviruses before the cells were continuously incubated for another 72 h. Three days after transfection, the transfection efficiency was examined by fluorescent microscopy.
8
Matrigel-Induced Tubulogenesis Assay
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Induction of tubulogenesis was performed using Matrigel. Matrigel was thawed on ice to prevent premature polymerization; aliquots of 50 μl were plated into individual wells of 96-well tissue culture plates (Costar) and allowed to polymerize at 37°C for at least 60 minutes. Cells were removed from confluent cultures by treatment with 0.05% trypsin, 0.53 mM EDTA. The cells were washed in serum-containing medium then resuspended to 106 cells/ml. Each culture well received 100-μl of the cell suspension with or without 100 μM TNF. Each control or TNF treatment was assayed in duplicate, and all experiments were performed at least three times. For quantitation of tube formation, the total length of the tubes formed in a unit area was digitized and measured using ECLIPSE software. For each test, five randomly chosen areas were measured and averaged.
9
Chicken Enterocyte Viability and Proliferation Assay
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Chicken enterocytes were harvested and cultured in Dulbecco's Modified Eagle Medium (DMEM ; catalog no. L0101-0500; VWR life science, NY) containing additional growth factor supplements (10% heat-inactivated fetal bovine serum [catalog no. 10082147; Thermo Fisher Scientific, Carlsbad, CA], IX insulin-transferrin-selenite [catalog no. I3146; Sigma-Aldrich], and 1X epithelial cell growth supplement [catalog no. PHG0313; Sigma-Aldrich]) as described previously (Rath et al., 2018 (link)). The effect of carvacrol on viability and proliferation of chicken enterocytes was determined using an MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] Cell Proliferation Assay Kit (ATCC 30-1010K; Manassas, VA) as per the standard published method (Zheng and Kunlong, 1992 ). Briefly, the monolayers of primary chicken enterocytes were grown in 96-well tissue culture plates (Costar) at ∼105 cells per well for 24 h. The enterocytes were treated with ethanol in DMEM (0.018%), carvacrol in DMEM (0.002%), or DMEM (control) for 2 h. The MTT reagent was added to each well, including blanks, and incubated at room temperature in the dark for 2 h. After the appearance of purple precipitation, detergent was added, and the absorbance was recorded at 570 nm by using a spectrophotometric microplate reader (Bio-Rad Laboratories, Hercules, CA).
10
Evaluating HGC Cytotoxicity in HepG2 Cells
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The cytotoxicity of HGC was
estimated using CCK-8. HepG2 cells were plated in 96-well tissue culture
plates (Corning Costar, NY, USA) at a density of 1 × 103 cells/well in MEM containing 10% fetal bovine serum and 1% P/S and
incubated for 1 day. After 24 h of incubation, the culture medium
was replaced with MEM containing various concentrations of HGC (0,
0.25, 0.5, and 1 wt %). After 0, 1, 3, 5, and 7 days of culture in
the medium with different concentrations of HGC, 10 μL of CCK
solution was directly added to each well, and the samples were incubated
at 37 °C for 2 h. An intense orange-colored and water-soluble
formazan derivative was formed via cellular metabolism in the culture
medium. The OD value for the culture medium was analyzed at a wavelength
of 450 nm using a microplate reader (VersaMax, Molecular Devices,
CA, USA). The relative proliferation rate was calculated by normalizing
data observed at each time point with data recorded at day 0.
estimated using CCK-8. HepG2 cells were plated in 96-well tissue culture
plates (Corning Costar, NY, USA) at a density of 1 × 103 cells/well in MEM containing 10% fetal bovine serum and 1% P/S and
incubated for 1 day. After 24 h of incubation, the culture medium
was replaced with MEM containing various concentrations of HGC (0,
0.25, 0.5, and 1 wt %). After 0, 1, 3, 5, and 7 days of culture in
the medium with different concentrations of HGC, 10 μL of CCK
solution was directly added to each well, and the samples were incubated
at 37 °C for 2 h. An intense orange-colored and water-soluble
formazan derivative was formed via cellular metabolism in the culture
medium. The OD value for the culture medium was analyzed at a wavelength
of 450 nm using a microplate reader (VersaMax, Molecular Devices,
CA, USA). The relative proliferation rate was calculated by normalizing
data observed at each time point with data recorded at day 0.
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