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96 well tissue culture plate

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The 96-well tissue culture plates are a commonly used laboratory equipment for cell culture applications. These plates provide a standardized multi-well format for culturing cells in a controlled environment. Each plate contains 96 individual wells, allowing for multiple samples or experimental conditions to be tested simultaneously. The plates are made of high-quality, tissue culture-treated polystyrene, providing a suitable surface for cell attachment and growth.

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Lab products found in correlation

Quanti blue substrate, by InvivoGen (6 mentions) Mst14, by R&D Systems (6 mentions) Epoch elisa reader, by Agilent Technologies (6 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions) Sytox green, by Thermo Fisher Scientific (3 mentions) Hek blue ifna b cells, by InvivoGen (3 mentions) Recombinant ifnα, by Novus Biologicals (3 mentions) Hek blue ifnα b cells, by InvivoGen (3 mentions) Recombinant ifna, by Novus Biologicals (3 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions) Roswell park memorial institute (rpmi), by Merck Group (2 mentions) Mtt solution, by Merck Group (2 mentions) Cell strainer, by BD (2 mentions) Concanavalin a (cona), by MP Biomedicals (2 mentions) Prism 5, by GraphPad (2 mentions) Synergy h1, by Agilent Technologies (2 mentions) Aim 5 albumax medium, by Thermo Fisher Scientific (2 mentions) Opti mem, by Thermo Fisher Scientific (2 mentions) Alamar blue, by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)

75 protocols using 96 well tissue culture plate

1

Lymph Node and Spleen Cell Culture

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The culture medium used was RPMI-1640 supplemented with 10% FCS, 100 μg/mL streptomycin, and 100 IU/mL penicillin. Cell suspensions were made by pressing the LNs through a cell strainer (Falcon, Franklin Lakes, NJ, USA). Cells were counted using a Coulter Counter. LN cell suspensions were cultured at 10
6cells/mL culture medium with 5 μg/mL Concanavalin A (MP Biomedicals, Irvine, CA, USA) in 96-well tissue culture plates (Nunc, Roskilde, Denmark) for 24 h. Spleen cell suspensions were cultured at 10
6cells/mL culture medium with 1 mg/mL OVA in 96-well tissue culture plates (Nunc) for 120 h. Culture conditions were 37 °C in a humidified atmosphere containing 5% CO
2.
Vandebriel R.J., Vermeulen J.P., van Engelen L.B., de Jong B., Verhagen L.M., de la Fonteyne-Blankestijn L.J., Hoonakker M.E, & de Jong W.H. (2018). The crystal structure of titanium dioxide nanoparticles influences immune activity in vitro and in vivo. Particle and Fibre Toxicology, 15, 9.
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2

Antiproliferative Effects of rfhSP-D on Prostate Cancer Cells

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Human PrEC (passage no. 3–5), LNCaP, PC3, or PrCEC (passage no. 3–5) cells (5 × 103) were placed in 96-well tissue culture plates (Nunc) and grown overnight. Cells were then starved in cell appropriate serum free media (PrEC and PrCEC for 4 h; LNCaP cells for 12 h; PC3 cells for 18 h) and treated with rfhSP-D (5, 10, and 20 μg/ml) for 24, 48, and 72 h. Cells alone in the culture medium served as an untreated control. After incubation 10 μl MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (5 mg/ml stock) was added to each well and incubated at 37°C for 4 h. Formazan crystals were dissolved in acidified iso-propanol and absorbance was read at 570 nm (Beckman Coulter).
Thakur G., Prakash G., Murthy V., Sable N., Menon S., Alrokayan S.H., Khan H.A., Murugaiah V., Bakshi G., Kishore U, & Madan T. (2019). Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells. Frontiers in Oncology, 9, 565.
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3

Viability of hBMECs in N. caninum Infection

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The nonradioactive colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl tetrazolium bromide (MTT, Sigma) reduction assay was used to determine the effect of N. caninum infection on the viability of hBMECs at 1, 3, 6, 12, 24, and 48 h post infection (hpi). Cultured hBMECs (104 cells/well) in 96-well tissue culture plates (Nunc) were incubated in 100 μL of RPMI medium for 18 h in a humidified incubator (37 °C, 5% CO2) until they became confluent. Tachyzoites of N. caninum were added, in triplicate, to hBMECs at MOI 2 for 2 h, followed by removal of the medium and washing with fresh RPMI medium to remove extracellular, non-attached, parasites. Each well was filled with 100 μL of fresh RPMI medium and the plates were incubated at the above culture conditions. Dimethyl sulfoxide (DMSO, 0.1%) was used as a control. At the indicated time points after infection, MTT was added to each well (to a final concentration of 0.5 mg/mL), and incubation was continued for a further 3–4 h in the dark at 37 °C. The cells were then incubated for 1 h in 100 µL of solubilizing solution (10% sodium dodecyl sulfate in 0.01 M HCl). The optical density (OD) was measured using a microtiter plate reader at 570nm. The experiment was performed in triplicate.
Elsheikha H.M., Alkurashi M., Palfreman S., Castellanos M., Kong K., Ning E., Elsaied N.A., Geraki K, & MacNaughtan W. (2020). Impact of Neospora caninum Infection on the Bioenergetics and Transcriptome of Cerebrovascular Endothelial Cells. Pathogens, 9(9), 710.
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4

Assessing Metabolic Activity of BM-SCs

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BM-SCs in a final concentration of 25 × 103 cells/mL were cultured in a volume of 200 µL of complete DMEM in 96-well tissue culture plates (Nunc, Roskilde, Denmark) for 48 h untreated or in the presence of tacrolimus, sirolimus, and mycophenolate mofetil (purchased from Sigma-Aldrich) in a concentration ranging from 0.005–500 µg/mL. The metabolic activity of the BM-SCs was determined using a WST-1 assay (Roche, Mannheim, Germany) and a Tecan Sunrise spectrophotometer (Life Science, Mannedorf, Switzerland), and analyzed by Kim 32 software (Schoeller Instruments, Prague, Czech Republic). The WST-1 solution (10 µL/100 µL of the sample) was added to samples for the final 3 h of the 48 h incubation period. The WST-1 is tetrazolium salt, which is cleaved to formazan through a cell mechanism, localized on a cell surface dependent on the production of NAD(P)H. Therefore, the formation of formazan correlates with the number of metabolically active cells.
Husakova J., Echalar B., Kossl J., Palacka K., Fejfarova V, & Dubsky M. (2023). The Effects of Immunosuppressive Drugs on the Characteristics and Functional Properties of Bone Marrow-Derived Stem Cells Isolated from Patients with Diabetes Mellitus and Peripheral Arterial Disease. Biomedicines, 11(7), 1872.
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5

TNF Bioactivity Assay Using WEHI 164 Cells

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Bioactive TNF was determined as previously described16 (link), using TNF sensitive fibroblast cell line WEHI 164, clone 13 (Walter and Eliza Hall Institute). WEHI cells were cultured in RPMI (Sigma, Germany) containing 10% FCS (Gibco, Invitrogen Corporation, Germany), 10 U/ml penicillin (Gibco, Germany), 10 μg/ml streptomycin (Gibco, Invitrogen Corporation, Germany) and 0.5× amino acid supplement (MEM Amino acids without L-glutamine, Gibco, Germany) at 37 °C with 5% CO2. Confluent cells were reseeded at 2 × 105 cells/ml in 96 well tissue culture plates (Nunclon, Denmark). TNF standards (recombinant mouse TNF, BD PharMingen, San Diego) in 2 fold serial dilutions or samples were added to WEHI cells and incubated for 18 h at 37 °C with 5% CO2. MTT solution (2 mg/ml, Sigma, Germany) was then added and incubated for a further 2 h, after which, the supernatants were aspirated and 50 μl DMSO was added and samples were read at 570 nm using VERSAmax Tunable Microplate Reader (Molecular devices Coorporation, California, USA). Data was analyzed using SoftMax Pro (Molecular devices Coorporation, California, USA).
Dambuza I.M., Keeton R., Hsu N.J., Allie N., Quesniaux V.F., Ryffel B, & Jacobs M. (2016). Persistent p55TNFR expression impairs T cell responses during chronic tuberculosis and promotes reactivation. Scientific Reports, 6, 39499.
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6

Cytotoxicity Assessment of Compounds

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The cytotoxic effect of compounds was determined using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit according to manufacturer's instructions (Promega Corporation, Madison, WI, USA). Compounds were diluted in DMSO to obtain 20 mM stock solutions. Appropriate aliquots of these stock solutions were further diluted in the cell culture medium to obtain final working concentrations. Cells (HuT78, MyLa, MJ, HH and PBMCs) were seeded at 5 × 104 cells per well in 96-well tissue culture plates (Nunc). Cells were treated with the various compounds at concentrations ranging from 1 to 100 μM for 24 h in triplicates. After the treatment period, cells were incubated with 20 μL MTS reagent (provided with the assay kit) for 2 h at 37 °C. Absorbance was recorded at 490 nm using a microplate reader (BioTek) and IC50 values for each compound were determined using GraphPad Prism (GraphPad Software Inc., La Jolla, CA, USA).
Verma N.K., Sadeer A., Kizhakeyil A., Pang J.H., Angela Chiu Q.Y., Tay S.W., Kumar P, & Pullarkat S.A. (2018). Screening of ferrocenyl–phosphines identifies a gold-coordinated derivative as a novel anticancer agent for hematological malignancies. RSC Advances, 8(51), 28960-28968.
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7

Semi-quantitative Biofilm Assay in 96-well Plates

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A semi-quantitative determination of biofilm formation was performed in 96-well tissue culture plates (Nunc, Rochester, NY, USA) based on the method reported by Christensen et al. (1985) [26 (link)]. Bacteria were grown in individual wells of 96-well plates at 37°C in tryptic soy broth (TSB; Becton Dickinson, NJ, USA) medium. After 24 h of growth, the plates were washed vigorously with 1X phosphate buffed saline (PBS), dried for 30 min at 55°C, and stained with 0.5% (w/v) crystal violet solution. After staining, the plates were washed with 1X PBS. A492 nm of the adhered, stained cells was measured using a Multiskan EX Microplate Photometer (Thermo Fisher Scientific, Lenexa, KS, USA). The criterion outlined by Chistensen et al. (1985) [26 (link)] was used to determine whether isolates were non-adherent and biofilm-negative (A492 < 0.12) or strongly biofilm-positive (A492 > 0.12). Assays were repeated six times, and the mean biofilm absorbance values were used. The results were analyzed using a one-way ANOVA with a Tukey’s test.
Flores-Páez L.A., Zenteno J.C., Alcántar-Curiel M.D., Vargas-Mendoza C.F., Rodríguez-Martínez S., Cancino-Diaz M.E., Jan-Roblero J, & Cancino-Diaz J.C. (2015). Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection. PLoS ONE, 10(8), e0135964.
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8

Cytotoxicity Evaluation of Gold Nanoparticles

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Cytotoxicity was determined using the MTT assay. On the day of exposure, exponentially growing cells were plated as 1×105 cells/well in 180 μL in 96-well tissue culture plates (Nunc). Cells were then incubated with medium (control), free doxorubicin, AuNPs B, and AuNPs D (20 μL, 10X in cell culture medium) for 2 h at 37 °C. Next the plate was centrifuged and supernatant was replaced with fresh medium (200 μL/well), which allowed for an additional 48 h of incubation. The viability was measured by quantifying the cellular ability to reduce the water-soluble tetrazolium dye 3–4,5-dimethylthiazole-2, 5-diphenyl tetrazolium bromide (MTT) to its insoluble formazan salt as follows. At 44 h, MTT (5 mg mL−1 in PBS, 20 μL/well) was added to the wells and the cells were further incubated for 4 h. At 48 h the supernatant was carefully removed and the water-insoluble formazan salt was dissolved in DMSO (120 μL/well). The absorbance was measured at 545 nm using a microplate reader (BMG Labtech). Data points were collected in triplicate and expressed as normalized values for untreated control cells (100%). Data were fitted using Prism software (GraphPad Software, Inc).
Hudlikar M.S., Li X., Gagarinov I.A., Kolishetti N., Wolfert M.A, & Boons G.J. (2015). Controlled Multi-functionalization Facilitates Targeted Delivery of Nanoparticles to Cancer Cells. Chemistry (Weinheim an der Bergstrasse, Germany), 22(4), 1415-1423.
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9

Competitive Binding Assay for US28 and CX3CR1

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Stable inducible clones of US28 and CX3CR1 cells were seeded at 10,000 cells/well in poly-D-lysine (Invitrogen) coated 96-well tissue culture plates (Nunc). One day after seeding US28 and CX3CR1 receptor expression was induced by tetracycline (Invitrogen; 3,6 ng/mL and 5 ng/mL, resp.) aimed at obtaining 5–10% specific binding of the added radioactive ligand. One day after induction, cells were assayed by competition binding for 3 h at 4°C using 20–70 pM 125I-CX3CL1 as well as unlabeled ligand 10 pM to 100 nM in 50 mM Hepes buffer pH 7.4, supplemented with 1 mM CaCL2, 5 mM MgCL2, and 0,5% (w/v) bovine serum albumin (BSA) (binding buffer). After incubation, cells were washed twice in ice-cold binding buffer and supplemented with 0,5 M NaCl. Determinations were made in quadruplicate.
Spiess K., Jeppesen M.G., Malmgaard-Clausen M., Krzywkowski K., Kledal T.N, & Rosenkilde M.M. (2017). Novel Chemokine-Based Immunotoxins for Potent and Selective Targeting of Cytomegalovirus Infected Cells. Journal of Immunology Research, 2017, 4069260.
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10

Cytotoxicity Assay of POE on A549 Cells

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Monolayers of human lung carcinoma-derived epithelial cells (A549, ATCC, CCL-185) were cultured as previously described [39 (link)]. Briefly, a detached cell suspension (5 × 105 cells/mL) was seeded at 100 µL/well into 96-well tissue culture plates (Nunc, Roskilde, Denmark) for 24 h at 37 °C. The culture medium was replaced with 100 µL medium with POE (3.9–4000 µg/mL) for 2 and 24 h, with appropriate positive and negative controls. The cytotoxicity was measured by MTT-reduction assay. The final absorbance (A550) of the samples was assessed using a microplate reader (Victor2, Wallac, Turku, Finland), and the percentage of viable cells and the IC50 were calculated.
Sadowska B., Wójcik U., Krzyżanowska-Kowalczyk J., Kowalczyk M., Stochmal A., Rywaniak J., Burzyńska J, & Różalska B. (2019). The Pros and Cons of Cystic Fibrosis (CF) Patient Use of Herbal Supplements Containing Pulmonaria officinalis L. Extract: the Evidence from an In Vitro Study on Staphylococcus aureus CF Clinical Isolates. Molecules, 24(6), 1151.
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