Polystyrene flat bottom plates

Manufactured by Corning

Sourced in United States

Polystyrene flat bottom plates are a type of lab equipment used for various applications in scientific research and laboratory settings. They are made of polystyrene, a common plastic material, and feature a flat bottom design. These plates are primarily used as containers or vessels for holding liquids, solutions, or samples during experiments and analyses.

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Lab products found in correlation

Ficoll paque, by GE Healthcare (5 mentions) Percoll, by Merck Group (3 mentions) Rpmi dutch modified medium, by Thermo Fisher Scientific (2 mentions) Rpmi medium, by Thermo Fisher Scientific (2 mentions) Pyruvate, by Thermo Fisher Scientific (1 mentions) Percoll, by Corning (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions)

7 protocols using polystyrene flat bottom plates

Isolation of Monocytes from Healthy Donors

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Buffy coats from healthy donors were obtained after written informed consent (Sanquin Blood Bank, Nijmegen, The Netherlands). Ethical approval was obtained from the CMO Arnhem-Nijmegen (NL32 357.091.10). Isolation was performed by differential density centrifugation over Ficoll-Paque (GE Healthcare, Chalfont St Giles, UK). Subsequently, isolation of monocytes was performed with a hyper-osmotic Percoll (Sigma-Aldrich, St Louis, MO, USA) density gradient centrifugation and washed once with pyrogen-free cold phosphate buffered saline (PBS). Cells were resuspended and later cultured in RPMI 1640 Dutch modified medium (Invitrogen, Waltham, MA, USA) supplemented with 5 μg/mL gentamicin (Centraform, Etten-Leur, the Netherlands), 2 mM Glutamax (Gibco, Walthan, MA, USA), and 1 mM pyruvate (Gibco). To ensure maximal purity, Percoll-isolated monocytes were left to adhere to polystyrene flat bottom plates (Corning, Sigma-Aldrish, New York, NY, USA) for 1 h at 37 °C 5% CO₂ and then washed once with warm PBS.

Ferreira A.V., Koeken V.A., Matzaraki V., Kostidis S., Alarcon-Barrera J.C., de Bree L.C., Moorlag S.J., Mourits V.P., Novakovic B., Giera M.A., Netea M.G, & Domínguez-Andrés J. (2021). Glutathione Metabolism Contributes to the Induction of Trained Immunity. Cells, 10(5), 971.

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Isolation and Purification of Monocytes

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PBMC isolation was performed by differential density centrifugation over Ficoll-Paque (GE Healthcare). Percoll isolation of monocytes was performed as previously described [46 (link)]. Briefly, 150–200 x 10⁶ PBMCs were layered on top of a hyper-osmotic Percoll solution and centrifuged for 15 min at 580g. The interphase layer was isolated and cells were washed with cold PBS. Cells were re-suspended in RPMI medium Dutch modified (Invitrogen) supplemented with 50 μg/mL gentamicin, 2 mM Glutamax, and 1 mM pyruvate, and counted. An extra purification step was added by adhering Percoll-isolated monocytes to polystyrene flat bottom plates (Corning) for 1 h at 37˚C; a washing step with warm PBS was then performed to yield maximal purity.

Tarancón R., Domínguez-Andrés J., Uranga S., Ferreira A.V., Groh L.A., Domenech M., González-Camacho F., Riksen N.P., Aguilo N., Yuste J., Martín C, & Netea M.G. (2020). New live attenuated tuberculosis vaccine MTBVAC induces trained immunity and confers protection against experimental lethal pneumonia. PLoS Pathogens, 16(4), e1008404.

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Isolation of Peripheral Blood Monocytes

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Buffy coats from healthy donors were obtained after written informed consent (Sanquin Blood Bank, Nijmegen, the Netherlands). Samples were anonymized to safeguard donor privacy. The use of the samples received IRB approval. Peripheral blood mononuclear cell (PBMC) isolation was performed by dilution of blood in pyrogen-free PBS and differential density centrifugation over Ficoll-Paque (GE Healthcare). Cells were washed three times in PBS. Percoll isolation of monocytes was performed as previously described (Repnik et al., 2003). Briefly, 150–200 x 10⁶ PBMCs were layered on top of a hyper-osmotic Percoll solution (48.5% Percoll [Sigma-Aldrich], 41.5% sterile H2O, and 0.16 M filter-sterilized NaCl) and centrifuged for 15 min at 580g. The interphase layer was isolated and cells were washed with cold PBS. Cells were re-suspended in RPMI culture medium (RPMI medium Dutch modified, Invitrogen) supplemented with 50 μg/mL gentamicin, 2 mM Glutamax, and 1 mM pyruvate, and counted. An extra purification step was added by adhering Percoll-isolated monocytes to polystyrene flat bottom plates (Corning) for 1 h at 37°C; a washing step with warm PBS was then performed to yield maximal purity.

Domínguez-Andrés J., Arts R.J., ter Horst R., Gresnigt M.S., Smeekens S.P., Ratter J.M., Lachmandas E., Boutens L., van de Veerdonk F.L., Joosten L.A., Notebaart R.A., Ardavín C, & Netea M.G. (2017). Rewiring monocyte glucose metabolism via C-type lectin signaling protects against disseminated candidiasis. PLoS Pathogens, 13(9), e1006632.

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Biofilm Formation Assay in 96-well Plates

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Biofilm formation in 96-well polystyrene plates was performed^{9 ,33 (link)}. S. aureus strains MR4 and MR23 and S. epidermidis strain SE21 were grown in BHI (2 mL) at 37 °C for 16–20 h. The cultures were diluted 1:1000 in BHIG and the suspensions (200-μL) were cultured at 37 °C for 24 h in 96-well polystyrene flat-bottom plates (Corning, Corning, NY). The resulting biofilms were used to examine the effects of clearing reagents on optical density and stability.

Sugimoto S, & Kinjo Y. (2023). Instantaneous Clearing of Biofilm (iCBiofilm): an optical approach to revisit bacterial and fungal biofilm imaging. Communications Biology, 6, 38.

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Isolation of Highly Purified Monocytes

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Buffy coats from healthy donors were obtained after written informed consent (Sanquin Blood Bank, Nijmegen, the Netherlands). Peripheral blood mononuclear cell (PBMC) isolation was performed by dilution of blood in pyrogen-free PBS and differential density centrifugation over Ficoll-Paque (GE Healthcare). Cells were washed twice in PBS. Training of adherent monocytes was performed as previously described (Quintin et al., 2012 (link)); see also below. Percoll isolation of monocytes was performed as previously described (Repnik et al., 2003 (link)). Briefly, 150 – 200 × 10⁶ PBMCs were layered on top of a hyper-osmotic Percoll solution (48.5% Percoll [Sigma-Aldrich], 41.5% sterile H₂O, and 0.16 M filter-sterilized NaCl) and centrifuged for 15 min at 580 × g. The interphase layer was isolated and cells were washed with cold PBS. Cells were resuspended in RPMI culture medium (RPMI medium, Invitrogen) supplemented with 10 μg/mL gentamicin, 10 mM Glutamax, and 10 mM pyruvate, and counted. An extra purification step was added by adhering Percoll-isolated monocytes to polystyrene flat bottom plates (Corning) for 1 hr at 37°C; a washing step with warm PBS was then performed to yield maximal purity.

Arts R.J., Novakovic B., ter Horst R., Carvalho A., Bekkering S., Lachmandas E., Rodrigues F., Silvestre R., Cheng S.C., Wang S.Y., Habibi E., Gonçalves L.G., Mesquita I., Cunha C., van Laarhoven A., van de Veerdonk F.L., Williams D.L., van der Meer J.W., Logie C., O’Neill L.A., Dinarello C.A., Riksen N.P., van Crevel R., Clish C., Notebaart R.A., Joosten L.A., Stunnenberg H.G., Xavier R.J, & Netea M.G. (2016). Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity. Cell metabolism, 24(6), 807-819.

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Trained Immunity in Monocyte-Derived Macrophages

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To increase the purity of Percoll‐isolated monocytes, the monocytes were adhered to polystyrene flat‐bottom plates (Corning) for 1 hr at 37° followed by washing with warm PBS. Next, cells were pre‐incubated with culture medium supplemented with 10% human pooled serum as control, or together with IgG1 isotype control or anti‐TLR10 antibody (10 μg/ml) for 1 hr. Subsequently, culture medium supplemented with 10% human pooled serum was added as a control, or together with either 2 μg/ml β‐glucan, or 5 μg/ml BCG. After 24 hr, cells were washed with warm PBS and culture medium was added. Culture medium was refreshed after 3 days of incubation. On day 6, cells were re‐stimulated with RPMI‐1640, lipopolysaccharide (LPS) (10 ng/ml), or Pam3Cys (10 μg/ml). After 24 hr, supernatants were collected and stored at −20° until further use (see Supplementary material, Fig. S1c). In the in vitro system used, monocytes differentiated into macrophages. However, the monocytes previously trained with β‐glucan or BCG undergo a different functional program characterized by increased responsiveness even long after the stimulus has been removed. This effect has been termed trained immunity.

Mourits V.P., Arts R.J., Novakovic B., Matzaraki V., de Bree L.C., Koeken V.A., Moorlag S.J., van Puffelen J.H., Groh L., van der Heijden C.D., Keating S.T., Netea M.G., Oosting M, & Joosten L.A. (2019). The role of Toll‐like receptor 10 in modulation of trained immunity. Immunology, 159(3), 289-297.

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Isolation and Purification of Monocytes

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Buffy coats (concentrated leukocyte suspensions obtained from whole human blood) from healthy donors were obtained after written informed consent (Sanquin Blood Bank, Nijmegen, Netherlands). Samples were anonymized to safeguard donor privacy. The peripheral blood mononuclear cell (PBMC) fraction consists of any peripheral blood cell having a round nucleus. These cells are lymphocytes (T cells, B cells, NK cells) and monocytes. Isolation of PBMCs was performed by differential density centrifugation over Ficoll-Paque (GE Healthcare). Percoll isolation of monocytes was performed as described in previous research (Repnik et al., 2003) . Briefly stated, 150-200 • 10 6 PBMCs were layered on top of a hyperosmotic Percoll solution and centrifuged for 15 min at 580 • g. The interphase layer was isolated, and cells were washed with cold phosphatebuffered saline (PBS). Cells were resuspended in RPMI medium with Dutch modification (Invitrogen) supplemented with 50 mg/mL gentamicin, 2 mM GlutaMAX, and 1 mM pyruvate, and counted. An additional purification step was added by adhering Percoll-isolated monocytes to polystyrene flat-bottom plates (Corning) for 1 h at 37°C. A washing step with warm PBS was then performed to yield maximal purity.

Domínguez-Andrés J., Eleveld M., Renieris G., Boltje T.J., Mesman R.J., van Niftrik L., Moons S.J., Rettberg P., van der Meer J.W.M., Giamarellos-Bourboulis E.J., Op den Camp H.J.M., de Jonge M.I, & Netea M.G. (2020). Growth on Carbohydrates from Carbonaceous Meteorites Alters the Immunogenicity of Environment-Derived Bacterial Pathogens. Astrobiology, 20(11).

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