96 well round bottom plate

Manufactured by BD

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The 96-well round-bottom plates are a type of laboratory equipment used for various assays and experiments. They provide a standardized platform with 96 individual wells, each with a rounded bottom shape. The plates are designed to facilitate the storage, mixing, and analysis of small liquid samples or reagents.

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96-well plates (481 citations) 96 well round bottom microtitre plates (3 citations) 96-well polypropylene round-bottom plates (2 citations)

56 protocols using 96 well round bottom plate

Peritoneal and Pleural Macrophage Activation

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On day prior to the experiment, cryopreserved peritoneal ascites from MSK 347c and pleural effusion from MSK 1366a was thawed and cultured in human complete RPMI media on a 10cm dish. On the day of the experiment, sodium metatungstate (50μM) was plated into respective wells of a 96 well round bottom plate (BD). After at least five rounds of gentle aspiration and washing of the 10cm plate containing the peritoneal lavage cells, adherent peritoneal macrophages were removed with a cell scraper, collected and counted. 10⁵ peritoneal or pleural macrophages were plated per indicated well on the 96 well plate. CD8⁺ T cells were purified from healthy donor PBMCs (NY Blood Center) utilizing the Human CD8⁺ T Cell Isolation Kit (EasySep) and then 10⁵ purified CD8⁺ T cells were plated in Human Complete Medium with 50IU/ml human IL-2. Certain wells were incubated with 10⁵ anti-human CD3/CD28 Dynabeads for T cell activation. Flow cytometry was performed after 48 hours of culture.

Chow A., Schad S., Green M.D., Hellmann M.D., Allaj V., Ceglia N., Zago G., Shah N.S., Sharma S.K., Mattar M., Chan J., Rizvi H., Zhong H., Liu C., Bykov Y., Zamarin D., Shi H., Budhu S., Wohlhieter C., Uddin F., Gupta A., Khodos I., Waninger J.J., Qin A., Markowitz G.J., Mittal V., Balachandran V., Durham J.N., Le D.T., Zou W., Shah S.P., McPherson A., Panageas K., Lewis J.S., Perry J.S., de Stanchina E., Sen T., Poirier J.T., Wolchok J.D., Rudin C.M, & Merghoub T. (2021). Tim-4+ Cavity-Resident Macrophages Impair Anti-Tumor CD8+ T cell Immunity. Cancer cell, 39(7), 973-988.e9.

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Hemagglutination Assay for Viral Titers

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To determine the hemagglutination unit (HAU) titer of each viral prep, viruses were diluted in a total volume of 50ul 2-fold across a 96-well round bottom plate (BD) and mixed with 12.5ul of 2% (vol/vol) turkey erythrocytes (Lampire) in PBS. Agglutination was read out after incubating for 60 minutes at room temperature.

Linderman S.L, & Hensley S.E. (2016). Antibodies with ‘Original Antigenic Sin’ Properties Are Valuable Components of Secondary Immune Responses to Influenza Viruses. PLoS Pathogens, 12(8), e1005806.

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Mechanism of IgA production in vitro

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To evaluate the mechanism for IgA production in vitro, B cells (5×10⁵) were either directly stimulated with LG2055 (10 µg/ml), BAFF (500 ng/ml, Sigma), TGF-β1 (0.001–1 ng/ml, R&D Systems), and LPS (10 µg/mL, InvivoGen) or co-cultured with CD11C⁺ cells derived from PP cells (0.5×10⁵) or BMDC (1×10⁵) in the presence or absence of LG2055 (10 µg/ml), LE135 (500 nM), SB505124 (5 µM), anti-human/mouse TLR2 (5 µg/ml), Pam3CSK4 (1 µg/mL, InvivoGen, CA), and FSL-1 (1 µg/mL, InvivoGen) in 96 well-round bottom plate (BD Bioscience) for 7 days. To block cell-to-cell contact, B cells (2×10⁶) and BMDC (4×10⁵) were cultured in 24 well culture plate (BD Bioscience) with or without a transwell device (BD Bioscience) in the presence or absence of LG2055 (10 µg/ml) for 7 days. The level of IgA in the culture supernatants was measured by mouse IgA ELISA.

Sakai F., Hosoya T., Ono-Ohmachi A., Ukibe K., Ogawa A., Moriya T., Kadooka Y., Shiozaki T., Nakagawa H., Nakayama Y, & Miyazaki T. (2014). Lactobacillus gasseri SBT2055 Induces TGF-β Expression in Dendritic Cells and Activates TLR2 Signal to Produce IgA in the Small Intestine. PLoS ONE, 9(8), e105370.

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Cytotoxic CD8+ T Cell Assay

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CD8⁺ T cells isolated from B16 tumor-bearing mice were cultured for 3 days with irradiated B16 stimulators in a complete 1640 RPMI medium. The responder cells were then collected and used as effector cells in a 8 h chromium release assay against indicated B16 target cells. B16 target cells were labeled by combining 5 × 10⁶ cells with 50 μCi 51Cr (Perkin-Elmer Japan Co.) in a total volume of 0.2 ml complete RPMI for 1 h at 37 °C, followed by washing thrice with plain RPMI. For the chromium release assay, CD8⁺ effector cells were mixed with B16 target cells at the different ratio of 1:1, 5:1 and 25:1 in a 96-well round-bottom plate (BD Biosciences).

Yang Z., Qi Y., Lai N., Zhang J., Chen Z., Liu M., Zhang W., Luo R, & Kang S. (2018). Notch1 signaling in melanoma cells promoted tumor-induced immunosuppression via upregulation of TGF-β1. Journal of Experimental & Clinical Cancer Research : CR, 37, 1.

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Peritoneal Macrophage-CD8+ T Cell Interaction

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On day prior to the experiment, peritoneal lavage cells were plated in complete RPMI media on a 10cm dish. Celexocib (10μM), anti-TGF-β (clone 1D11, 10μg/ml), or sodium metatungstate (50μM) was plated onto respective wells of a 96 well round bottom plate (BD). After at least five rounds of gentle aspiration and washing of the 10cm plate containing the peritoneal lavage cells, adherent peritoneal macrophages were removed with a cell scraper, collected and counted. 10⁵ peritoneal macrophages were plated per indicated well of the 96 well plate. CD8⁺ T cells were purified from ACK-lysed splenocytes from C57BL/6 mice utilizing the Mouse CD8a+ T Cell Isolation Kit and then 10⁵ purified CD8⁺ T cells were plated onto in T cell medium with 50IU/ml murine IL-2. Certain wells were incubated with 10⁵ anti-mouse CD3/CD28 Dynabeads for T cell activation. Flow cytometry was performed after 48 hours of culture.

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Proliferation Assay of Dendritic Cells

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Cell proliferation assays were determined by BrDu (colorimetric assay kit) assays according to manufacturer protocol (Sigma Aldrich). Briefly, mDCs with or without 5 μg/mL of HSP60 were cultured for 12 hours. After washing and resuspending in complete RPMI media, 0.5×10⁵ DCs were cocultured with 2×10⁵ autologous T cells in a 96‐well round‐bottom plate (Becton Dickinson). After 72 hours of incubation, cells were labeled with BrDu and incubated for 20 hours. Cells were collected, centrifuged, and dried at 60°C for 1 hour. Dried cells were fixed with FixDenat solution before incubation with anti‐BrDu POD (Peroxidase) antibodies. After 2 hours of incubation, cells were washed and substrate solution was added for color development. To stop the reaction, 1M H₂SO₄ was added into substrate solution and read the reaction plate at 450‐nm wavelength with 690 reference wavelength.

Rahman M., Steuer J., Gillgren P., Hayderi A., Liu A, & Frostegård J. (2017). Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(11), e006778.

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Whole Blood TLRA Activity Assay

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For assessment of TLRA activity in whole blood, we used an adaptation of the method of Kollmann et al [37 (link)]. Neonatal cord blood, infant peripheral blood, or adult peripheral blood was diluted 5x with sterile pre-warmed (37°C) RPMI 1640 medium (Invitrogen, Carlsbad, CA) and 135 μL of the suspension was added to each well of a 96 well round-bottom plate (Becton Dickinson, Franklin Lakes, NJ, USA) containing 15 μl freshly prepared specific TLRAs at 10x the final concentration. Suspensions containing 150 μl/well were gently mixed by pipetting and incubated for 18 h at 37°C in a humidified incubator at 5% CO₂. After culture, plates were centrifuged at 500 × g and 3 aliquots of 35 μl of supernatant were carefully removed by pipetting without disturbing the cell pellet. Supernatants derived from human leukocyte stimulations were assayed by ELISA for TNF (BD Biosciences, San Jose, CA, USA) and PGE₂ (Cayman Chemicals; Ann Arbor, MI) and by multiplexing bead array (Millipore).

Van Haren S.D., Ganapathi L., Bergelson I., Dowling D.J., Banks M., Samuels R.C., Reed S.G., Marshall J.D, & Levy O. (2016). In vitro cytokine induction by TLR-activating vaccine adjuvants in human blood varies by age and adjuvant. Cytokine, 83, 99-109.

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Cell Proliferation Assay of Dendritic Cells

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Cell proliferation was determined using the colorimetric BrdU kit in accordance with the manufacturer's protocol (Sigma Aldrich). In brief, DCs cultured with or without 10 μg/ml MDA-HSA were cultured for 12 h, washed, and re-suspended in the complete RPMI medium, and then 0.5 × 10⁵ DCs were co-cultured with 2 × 10⁵ autologous T cells in a 96-well round-bottom plate (Becton Dickinson, Franklin Lakes, New Jersey). In addition, T cells were also cultured alone with or without MDA-HSA. After 72 h of incubation, the cells were labeled with BrdU, incubated for 20 more hours, centrifuged, and dried at 60 °C for 1 h. The dried cells were fixed with a FixDenat (Roche) solution before incubation with anti-BrdU peroxidase antibodies. After 2 h of incubation, the cells were washed and substrate solution added for development of color. To stop the reaction, 1 mol/l H₂SO₄ was added and absorption at 450-nm wavelength (with 690 nm as reference) determined.

Rahman M., Steuer J., Gillgren P., Végvári Á., Liu A, & Frostegård J. (2019). Malondialdehyde Conjugated With Albumin Induces Pro-Inflammatory Activation of T Cells Isolated From Human Atherosclerotic Plaques Both Directly and Via Dendritic Cell–Mediated Mechanism. JACC: Basic to Translational Science, 4(4), 480-494.

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Endoxifen Sensitivity Profiling in LCLs

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We have previously developed a method for phenotyping cellular sensitivity to endoxifen in HapMap LCLs [10] . This method was applied to evaluate cellular sensitivity to endoxifen in 58 unrelated HapMap YRI LCLs. Specifically, each HapMap LCL sample was transferred into “hormone-free” media (phenol red free RPMI1640, 15% charcoal stripped FBS, and 1% L-glutamine) 72 hours prior to the experiment. The LCLs that displayed >85% viability were pelleted and resuspended in the new “hormone-free” media at a concentration of 350,000 cells/ml. These LCLs were plated at a concentration of 10,000 cells/well in hormone-free media in triplicate in a 96-well round bottom plate (Becton Dickinson Labware, Franklin Lakes, NJ). Twenty-four hours later, increasing concentrations of endoxifen (3, 5, 7, 10 μM) were added to these LCLs, incubating for another 72 hours. AlamarBlue (Biosource, Camarillo, CA) was added 24 hours before growth inhibition evaluation using the Synergy-HT multi-detection plate reader (BioTek, Winooski, VT). The percent survival at each treatment concentration compared to control was obtained for each cell line via the manufacturer’s protocol [12] (link).

Weng L., Ziliak D., LaCroix B., Geeleher P, & Huang R.S. (2014). Integrative “Omic” Analysis for Tamoxifen Sensitivity through Cell Based Models. PLoS ONE, 9(4), e93420.

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Evaluating HDAC6 Inhibitor's Impact on Treg-Mediated Teff Cell Suppression

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Treg cells were mixed with eFluor®670-labeled Teff cells at ratios of 4:1 and 0:1 in RPMI1640 media (Gibco, MA, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco), 1% penicillin/streptomycin (P/S; Gibco), 1 mM sodium pyruvate (Gibco), and 1% 2-mercaptoethanol (Gibco) in a 96-well round bottom plate (Becton Dickinson). The mixed cells were incubated with vehicle (DMSO final concentration 0.1%) or HDAC6 inhibitor (1 to 10 uM) in the presence of antiCD3/CD28 beads (T cell Activation/Expansion kit, Miltenyi Biotec) for 4 days. Proliferation of Teff cells was analyzed using FACS LSR Fortessa (BD bioscience). The results were analyzed by FlowJo software (TreeStar Inc.).

Oh B.R., Suh D.H., Bae D., Ha N., Choi Y.I., Yoo H.J., Park J.K., Lee E.Y., Lee E.B, & Song Y.W. (2017). Therapeutic effect of a novel histone deacetylase 6 inhibitor, CKD-L, on collagen-induced arthritis in vivo and regulatory T cells in rheumatoid arthritis in vitro. Arthritis Research & Therapy, 19, 154.

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