Round bottom 96 wells plates

Manufactured by Greiner

Round-bottom 96-wells plates are a standard laboratory equipment used to hold and process small liquid samples. These plates feature a round-bottom well design and are typically made of polystyrene or polypropylene materials. They are available in various sizes and can be used for a variety of applications in life science research and diagnostics.

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Lab products found in correlation

Escherichia coli lps, by Merck Group (2 mentions) Ficoll paque, by GE Healthcare (2 mentions) Macs microbead, by Miltenyi Biotec (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Lipopolysaccharides (lps), by Merck Group (1 mentions) Rhtnf α, by Thermo Fisher Scientific (1 mentions) Rhil 15, by Thermo Fisher Scientific (1 mentions) Rhil 18, by R&D Systems (1 mentions) Rhil 6, by Miltenyi Biotec (1 mentions) Recombinant human il 18, by R&D Systems (1 mentions)

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96-well plates (620 citations) Round-bottom 96-well plates (61 citations) 96-well clear round-bottom plates (2 citations) Round bottom 96-well micro plates (2 citations)

6 protocols using round bottom 96 wells plates

PBMC Stimulation Assay Protocol

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PBMC stimulation experiments were performed within 6 hours after drawing of blood. PBMC stimulation experiments were performed on round bottom 96 wells plates (Greiner). In each well, 100 μL of PBMC cell suspension (5×10⁶/mL) was seeded. Subsequently, 100 μL of stimulus was added. The final cell culture concentrations of the used stimuli were as follows: 10 ng/mL Escherichia coli LPS (serotype 055:B5, Sigma-Aldrich), 10 µg/mL Pam-3-Cys (EMC microcollection). The experiments were performed in duplicate. PBMCs were stimulated for 24 hours in an incubator at 37°C and 5% CO2. After 24 hours the plates were spun for 8 min at 1400 RPM (Rotina 380R Hettich) and supernatants were collected and stored at −80°C until analysis.

van Puffelen J.H., Novakovic B., van Emst L., Kooper D., Zuiverloon T.C., Oldenhof U.T., Witjes J.A., Galesloot T.E., Vrieling A., Aben K.K., Kiemeney L.A., Oosterwijk E., Netea M.G., Boormans J.L., van der Heijden A.G., Joosten L.A, & Vermeulen S.H. (2023). Intravesical BCG in patients with non-muscle invasive bladder cancer induces trained immunity and decreases respiratory infections. Journal for Immunotherapy of Cancer, 11(1), e005518.

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Isolation and Stimulation of PBMCs

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Isolation of PBMCs was performed as described in Oosting et al. (Oosting et al., 2015 (link)). Cells were washed twice in saline and suspended in medium (RPMI 1640) supplemented with gentamicin 10 mg/mL, L-glutamine 10 mM and pyruvate 10 mM. PBMC stimulations were performed with 5×10⁵ cells/well in round-bottom 96-wells plates (Greiner) for either 24 hours or 7 days in the presence of 10% human pool serum at 37°C and 5% CO₂. Additional details are available in the Supplemental Information. Supernatants were collected and stored in −20°C until used for ELISA. The stimulations used for the 24 hour and 7 day experiments are shown in Table S1.

ter Horst R., Jaeger M., Smeekens S.P., Oosting M., Swertz M.A., Li Y., Kumar V., Diavatopoulos D.A., Jansen A.F., Lemmers H., Toenhake-Dijkstra H., van Herwaarden A.E., Janssen M., van der Molen R.G., Joosten I., Sweep F.C., Smit J.W., Netea-Maier R.T., Koenders M.M., Xavier R.J., van der Meer J.W., Dinarello C.A., Pavelka N., Wijmenga C., Notebaart R.A., Joosten L.A, & Netea M.G. (2016). Host and environmental factors influencing individual human cytokine responses. Cell, 167(4), 1111-1124.e13.

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Isolation and Stimulation of Immune Cells

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The mononuclear cell fraction was isolated from blood by density centrifugation of blood, diluted 1:1 in pyrogen-free saline over Ficoll-Paque (Pharmacia Biotech, PA, USA). Cells were washed twice in saline and resuspended in culture medium (RPMI, Invitrogen, CA, USA) supplemented with gentamicin 10 μg/ml, L-glutamine 10 mM, and pyruvate 10 mM. CD14+ (monocytes) and CD56+ (NK cells) subsets were purified from freshly isolated PBMCs using MACS microbeads, according to the instructions of the manufacturer (Miltenyi Biotec). Purity was checked with FACS and was >90%. Cells were counted in a Coulter counter (Coulter Electronics) and the number was adjusted to 5×10⁵ cells/ml. A total of 1 × 10⁵ monocytes or NK cells in a 100μl volume was added to round-bottom 96-wells plates (Greiner) with RPMI (with a additions as previously mentioned) or with sonicated MTB H37Rv (1μg/ml final concentration), heat-killed Candida albicans (1×10⁶ microorganisms/ml, strain UC820), Staphylococcus aureus (1×10⁶ microorganisms/ml), or E. coli lipopolysaccharide 1ng/ml (LPS, Sigma-Aldrich, 1ng/ml). After 48 hours supernatants were stored at −20°C. Cytokine concentrations were assessed in the supernatants using enzyme-linked immunosorbent assay (ELISA).

Kleinnijenhuis J., Quintin J., Preijers F., Joosten L.A., Jacobs C., Xavier R.J., van der Meer J.W., van Crevel R, & Netea M.G. (2014). BCG-induced trained immunity in NK cells: role for non-specific protection to infection. Clinical immunology (Orlando, Fla.), 155(2), 213-219.

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Isolation and Stimulation of PBMCs

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After obtaining informed consent, venous blood was drawn from the cubital vein of volunteers into 10 mL EDTA tubes (Monoject). Isolation of PBMCs was performed according standard protocols, with minor modifications. The PBMC fraction was obtained by density centrifugation of blood diluted 1:1 in pyrogen-free saline over Ficoll-Paque (Pharmacia Biotech). Cells were washed twice in saline and suspended in medium (RPMI 1640) supplemented with gentamicin 10 mg/mL, L-glutamine 10 mM and pyruvate 10 mM. Addition of antibiotics such as gentamycin is a standard methodology used in order to avoid contamination of cultures, and it does not influence the ability to induce cytokine production by PBMCs or macrophages (data not shown). The cells were counted in a Coulter counter (Coulter Electronics) and the number was adjusted to 5×10⁶ cells/mL. Then 5×10⁵ PBMCs in a 100 μL volume were added to round-bottom 96-wells plates (Greiner) and incubated with 100 μL of stimulus. After 24 h the supernatants were collected and stored at −20°C until assayed. The stimulation time periods were chosen based on extensive previous studies that showed that 24 h stimulation was best suited to assess monocyte-derived cytokines^{42 (link),43 (link)}.

Li Y., Oosting M., Deelen P., Ricaño-Ponce I., Smeekens S., Jaeger M., Matzaraki V., Swertz M.A., Xavier R.J., Franke L., Wijmenga C., Joosten L.A., Kumar V, & Netea M.G. (2016). Inter-individual variability and genetic influences on cytokine responses against bacterial and fungal pathogens. Nature medicine, 22(8), 952-960.

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Stimulation of NK Cells with B. pertussis

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NK cells were seeded into round-bottom 96-wells plates (Greiner) at 150,000 cells/well in IMDM culture medium supplemented with 5 ng/ml recombinant human IL-15 (rhIL-15; PeproTech) (29 (link)). Next, NK cells were immediately stimulated with B. pertussis B4393 at a MOI of 10 in the presence or absence of 5 ng/ml recombinant human IL-18 (rhIL-18; R&D systems), 10 ng/ml rhIL-6 (Miltenyi Biotec), 10 ng/ml rhTNFα (PeproTech), or 10 ng/ml rhIL-1β (InvivoGen). Stimulations were performed at 37°C and 5% CO₂ for 18 h after which BD GolgiPlug^TM containing Brefeldin A (BD Biosciences) was added to the culture for 4 h, to inhibit cytokine secretion, before collecting the NK cells for flow cytometry analysis.

Kroes M.M., Mariman R., Hijdra D., Hamstra H.J., van Boxtel K.J., van Putten J.P., de Wit J, & Pinelli E. (2019). Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages. Frontiers in Immunology, 10, 2030.

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PBMC Stimulation and Cytokine Profiling

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PBMC stimulation experiments were performed within 6 hours after drawing of blood. PBMC stimulation experiments were performed on round bottom 96 wells plates (Greiner). In each well 100uL of PBMC cell suspension (5x10 6 /mL) was seeded. Subsequently, 100uL of stimulus was added. The final cell culture concentrations of the used stimuli were as follows: 10 ng/mL Escherichia coli LPS (serotype 055:B5, Sigma-Aldrich), 10 µg/mL Pam-3-Cys (EMC microcollection). The experiments were performed in duplicate. PBMCs were stimulated for 24 hours in an incubator at 37°C and 5% CO2. After 24 hours the plates were spun for 8 minutes at 1400RPM (Rotina 380R Hettich) and supernatants were collected and stored at -80°C until analysis.

Puffelen J.H.v., Novakovic B., Emst L.v., Kooper D., Zuiverloon T.C., Oldenhof U.T., Witjes J., Galesloot T.E., Vrieling A., Aben K.K., Kiemeney L.A., Oosterwijk E., Netea M.G., Boormans J.L., Heijden A.G.v.d., Joosten L.A.B., & Vermeulen S.H. (2022). Intravesical BCG in patients with non-muscle invasive bladder cancer induces trained immunity and decreases respiratory infections.

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