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Six well plates

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Six-well plates are a type of laboratory equipment used for cell culture and various other experimental applications. These plates feature six individual wells arranged in a 2x3 grid pattern, providing a standardized and organized platform for culturing or processing multiple samples simultaneously.

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Lab products found in correlation

Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Methyl thiazolyl tetrazolium (mtt), by Thermo Fisher Scientific (1 mentions) 96 well culture plate, by Merck Group (1 mentions) Rhvegf, by Merck Group (1 mentions) Trypan blue stain, by Thermo Fisher Scientific (1 mentions) Sodium pyruvate, by Thermo Fisher Scientific (1 mentions) Human pan monocyte isolation kit, by Miltenyi Biotec (1 mentions) Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Ficoll paque premium, by GE Healthcare (1 mentions) Ls column, by Miltenyi Biotec (1 mentions) Fetal bovine serum (fbs), by Gemini Bio (1 mentions) Penicillin streptomycin, by Corning (1 mentions) Tc20 automated cell counter, by Bio-Rad (1 mentions) Cytarabine, by Merck Group (1 mentions) Daunorubicin, by Selleck Chemicals (1 mentions) Etoposide, by Merck Group (1 mentions) Irdye 800 goat anti rabbit antibody, by LI COR (1 mentions) Phospho p53 antibody sampler kit, by Cell Signaling Technology (1 mentions)

10 protocols using six well plates

1

VEGF-Induced Endothelial Progenitor Cell Proliferation

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The proliferation assay was performed as previously described [12 (link)]. Briefly, EPCs were digested with 0.25% trypsin (Sagene) and then seeded in 96-well culture plates (Sigma). After overnight culture in phenol red-free DMEM, cells were incubated with 100 ng/mL rhVEGF (ProSpec, East Brunswick, NJ, USA) for 24 hours. Before the optical density measurement (562 nm, Tecan, Zurich, Switzerland) was performed, EPCs were supplemented with 10 μL MTT (5 g/L, Gibco) for another six hours and 200 μL DMSO for 10 minutes.
Mitogenic activity was further evaluated by cell counting. EPCs were seeded in six-well plates (Sigma) at a density of 5 × 105 cells per well in phenol red-free DMEM overnight, and then exposed to 100 ng/mL rhVEGF for another 24 hours.
Yin Y., Liu H., Wang F., Li L., Deng M., Huang L, & Zhao X. (2015). Transplantation of cryopreserved human umbilical cord blood-derived endothelial progenitor cells induces recovery of carotid artery injury in nude rats. Stem Cell Research & Therapy, 6(1), 37.
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2

Tachyzoite Plaque Assay in HFFs

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Plaque assays were performed by inoculating 100 tachyzoites onto confluent monolayers of HFFs grown in six-well plates (Sigma-Aldrich). Cells were left undisturbed for 7–9 d, and monolayers were then fixed in 80% EtOH and stained with violet (Sigma-Aldrich). Plaque number and size were quantitated using ImageJ software and analysed in Prism (GraphPad). Samples were tested for normal distribution before statistical testing.
Wilde M.L., Ruparel U., Klemm T., Lee V.V., Calleja D.J., Komander D, & Tonkin C.J. (2023). Characterisation of the OTU domain deubiquitinase complement of Toxoplasma gondii. Life Science Alliance, 6(6), e202201710.
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3

Differentiation of Human Macrophages from PBMCs

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Human macrophages were differentiated from peripheral blood mononuclear cells (PBMCs) as described in Noel et al.93 (link). Briefly, human blood was obtained from healthy adult volunteers. PBMCs were purified from 10 ml of EDTA-treated human blood using Ficoll-Paque PREMIUM density 1.007 g/ml (GE Healthcare). Contaminating red blood cells were lysed with ACK lysis buffer for 5 min at room temperature (RT). Monocytes were enriched by negative selection using the human Pan Monocyte Isolation Kit (Miltenyibiotec) and LS Columns (Miltenyibiotec). Monocytes were resuspended in RPMI supplemented with 10% FBS (Gemini Bio-products), 55 μM 2-Mercaptoethanol (Gibco), 1 mM Sodium Pyruvate (Gibco), 1x MEM non-essential amino acids (Gibco), and 1x penicillin/streptomycin (Corning). Cells were counted using Trypan blue stain (0.4%; Thermo Fisher Scientific) in a TC20™ Automated cell counter (Bio-Rad) and 2 × 106 monocytes were seeded into six-well plates (Sigma). Human recombinant Macrophage Colony-Stimulating Factor (M-CSF; Biolegend) was added to each well at 50 ηg/ml final concentration on days 0, 2, and 4 after seeding. Medium was changed on day 4. Cells were incubated at 37 °C 5% CO2 for 6 days to allow differentiation into M0 macrophages.
Oliva Chávez A.S., Wang X., Marnin L., Archer N.K., Hammond H.L., Carroll E.E., Shaw D.K., Tully B.G., Buskirk A.D., Ford S.L., Butler L.R., Shahi P., Morozova K., Clement C.C., Lawres L., Neal A.J., Mamoun C.B., Mason K.L., Hobbs B.E., Scoles G.A., Barry E.M., Sonenshine D.E., Pal U., Valenzuela J.G., Sztein M.B., Pasetti M.F., Levin M.L., Kotsyfakis M., Jay S.M., Huntley J.F., Miller L.S., Santambrogio L, & Pedra J.H. (2021). Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection. Nature Communications, 12, 3696.
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4

Assessing Nanoparticle Cytotoxicity in Cell Lines

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To ensure the concentrations used were non‐toxic, a fixed number of cells were seeded onto six‐well plates (Sigma‐Aldrich, UK), one plate for each cell line, where two concentrations of each nanoparticle were introduced. From the literature, it was reported that a concentration of 0.5 mg mL−1 (24 h incubation) was not toxic; therefore, this and a lower concentration of 0.2 mg mL−1 were tested.[18 (link)
] Following the incubation period, medium and nanoparticle mixture was removed, wells were gently washed twice with PBS, and each well was filled with 5 mL of fresh medium. The plates were incubated for 10–14 days, fixed, stained, and colonies were counted and compared to control plates without NPs.
Ahmad R., Schettino G., Royle G., Barry M., Pankhurst Q.A., Tillement O., Russell B, & Ricketts K. (2020). Radiobiological Implications of Nanoparticles Following Radiation Treatment. Particle & Particle Systems Characterization, 37(4), 1900411.
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5

Co-culture of Leukemia Cells with BMSCs

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For co‐culture conditions, six‐well plates (Sigma) were coated with 0.1% gelatin (Sigma) in PBS for 20 minutes at 37°C. Gelatin solution was removed and BMSCs were seeded at a density of 2 × 105 cells/well and cultured overnight until a confluent monolayer was obtained. Subsequently, 2.5 × 104 HL‐60, Kasumi‐1 and OCI/AML3 cells were plated per well. The cultures were treated with or without 10−6 mol/L cytarabine (Sigma) or 10−7 mol/L daunorubicin (Selleckchem, Houston, TX) as well as 10−6 mol/L etoposide (Sigma) for 72 hours.
Su M., Chang Y., Hernandez D., Jones R.J, & Ghiaur G. (2019). Regulation of drug metabolizing enzymes in the leukaemic bone marrow microenvironment. Journal of Cellular and Molecular Medicine, 23(6), 4111-4117.
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6

Phospho-p53 Immunostaining Protocol

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Cells were seeded at a density of 1 × 106 cells onto in six-well plates (Sigma-Aldrich) and incubated with 100 µM PT, OT and AM as above. After the incubation, the cells were washed with ice-cold PBS and processed as described by us13 (link). Immunostaining of phosphorylated p53 at Ser9, Ser20, and Ser392 on nitrocellulose membranes (1:1000, GE Healthcare) was performed using the phospho-p53 antibody sampler kit (Cell Signaling Technology, 9919) followed by secondary detection using IRDye-800 goat anti-rabbit antibody (1:5000, Licor Biosciences, 925–32211) and visualized using the Licor Odyssey Imaging system (Licor Biosciences).
Chornyy S., Parkhomenko Y, & Chorna N. (2017). Thiamine antagonists trigger p53-dependent apoptosis in differentiated SH-SY5Y cells. Scientific Reports, 7, 10632.
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7

RNA Isolation and Purification Protocol

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Cells were seeded at a density of 1 × 106 cells onto in six-well plates (Sigma-Aldrich) and incubated with 100 µM PT, OT and AM as above. Total RNA was isolated using the TRIZOL® reagent (Thermofisher Sci) followed by DNase I treatment (Quiagen) and processed as described by us13 (link). The concentration and integrity of RNA were determined using the NanoDrop-1000 Spectrophotometer (Thermofisher Sci).
Chornyy S., Parkhomenko Y, & Chorna N. (2017). Thiamine antagonists trigger p53-dependent apoptosis in differentiated SH-SY5Y cells. Scientific Reports, 7, 10632.
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8

Quantifying Anti-HisRS IgG Antibodies

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Anti-HisRS IgG antibodies in the serum were measured using standard solid-phase enzyme-linked immunosorbent assay (ELISA) as described [11 (link), 12 (link)]. Briefly ninety-six-well plates (Sigma) were coated with recombinant murine synthetic HisRS N-terminal fragment sequence (0.1 μg/mL) and incubated overnight at 4°C. After blocking with PBS containing 1% bovine serum albumin (BSA), serum at various dilutions in PBS containing 1% BSA was added for 2 hours. Following incubation with HRP-conjugated goat anti-mouse IgG (Sigma), the enzymatic reaction was visualized using 3,3′,5,5′-tetramethylbenzidine (Sigma) and terminated by H2SO4 addiction prior to spectrophotometric assessment at 450 nm using a microplate reader (Biorad, Hercules, CA) [11 (link), 12 (link)].
Sciorati C., Esposito A., Campana L., Canu T., Monno A., Palmisano A., De Cobelli F., Del Maschio A., Ascheman D.P., Manfredi A.A, & Rovere-Querini P. (2014). 7-Tesla Magnetic Resonance Imaging Precisely and Noninvasively Reflects Inflammation and Remodeling of the Skeletal Muscle in a Mouse Model of Antisynthetase Syndrome. BioMed Research International, 2014, 879703.
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9

GR Activation and Reporter Assay

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Luciferase assays were performed using six-well plates (9.4 cm 2 wells, Sigma), except where noted. The NEB Gaussia and Cypridina plasmids were used at 400 ng each per well for each six-well assay (or 10 times less for 96-well plates). Two GR response elements were previously cloned into the promoter of the Gaussia plasmid, and the Cypridina plasmid served as a control. (4) GR expression plasmids were transfected at a level of 6 ng per well, and TSG101 plasmids were transfected from 0 to 94 ng per well, with the empty pJ603 plasmid balancing the total transfection to 100 ng of pJ603 DNA. Gaussia and Cypridina substrates were obtained from NEB or its original supplier, Targeting Systems. Either a Berthold or a Promega luminometer were used for automated data collection.
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White J.T., Rives J., Tharp M.E., Wrabl J.O., Thompson E.B, & Hilser V.J. (2021). Tumor Susceptibility Gene 101 Regulates the Glucocorticoid Receptor through Disorder-Mediated Allostery. Biochemistry, 60(21).
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10

Culturing Melanoma Lymph Node Cells

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MNC isolated from regional LNs of melanoma patients were cultivated for 7 days in RPMI 1640 culture medium (CM) alone, CM supplemented with IL-2 (200 U/ml) and IL-15 (25 ng/ml) in six-well plates (Sigma-Aldrich, Germany) at 37 0 C and 5% CO 2 in humid atmosphere. After 3 days of in vitro cultivation CM, as well as CM supplemented with these cytokines in proper concentrations, were added.
Vuletić A., Jovanić I., Jurišić V., Milovanović Z., Nikolić S., Spurnić I, & Konjević G. (2020). IL-2 And IL-15 Induced NKG2D, CD158a and CD158b Expression on T, NKT- like and NK Cell Lymphocyte Subsets from Regional Lymph Nodes of Melanoma Patients. Pathology oncology research : POR, 26(1).
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