El406 plate washer

Manufactured by Agilent Technologies

The EL406 plate washer is a laboratory instrument designed for automated washing of microplates. It is capable of performing precise and consistent washing of well-based assays, such as ELISA, cell-based, and other types of microplate-based assays. The EL406 plate washer utilizes a patented manifold design to deliver accurate liquid dispense and aspiration, ensuring efficient removal of unwanted materials from the microplate wells.

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EL406 (40 citations) Plate washer (36 citations) EL406 washer (4 citations) EL406 plate washer/dispenser (4 citations) EL406 plate washer and dispenser (2 citations)

11 protocols using el406 plate washer

High-Throughput Screening of Selleck Cambridge Cancer Library

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The Selleck Cambridge Cancer Library was provided and screened by the KI HTS Facility. The complete list of compounds is available in Supplementary Information Table 4. All compounds were used at single dose, final concentration of 10 μM. A total of 392 compounds were arrayed in 96-well plates, 80 compounds per plate and were tested in duplicate assay plates. Each plate contained 8 negative control DMSO wells. One plate was not treated with MG132 and used as positive control (no stress). Drugs were added to cell assay plates using a 250 nl pin transfer tool (V&P Scientific) mounted on the MCA96 head of a Freedom Evo 150 (Tecan) liquid handler. Cell staining was performed using a Biotek EL406 plate washer.

Gaglia G., Rashid R., Yapp C., Joshi G.N., Li C.G., Lindquist S.L., Sarosiek K.A., Whitesell L., Sorger P.K, & Santagata S. (2020). HSF1 phase transition mediates stress adaptation and cell fate decisions. Nature cell biology, 22(2), 151-158.

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Transfection and Viral Infection Assay Protocol

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Transfection with siRNAs was performed according to manufacturers’ instructions using RNAiMAX (Thermo Fisher Scientific). We performed reverse transfection by seeding 80 μl HeLa cells (2 × 10³ cells/well) onto a 20 μl mix of siRNA (10 nM) and transfection reagent. Hela MZ cells were infected with VSV 19 using a BioTek™ EL406 plate washer, fixed with 4% PFA and stained with DAPI, and images were acquired with the IXM™ microscope and analysed with MetaXpress™ to quantify infected cells. The cholesterol content of liver extracts was measured enzymatically using the Amplex Red Kit (Molecular Probes). Dried lipid samples were re‐dissolved in pure methanol, sonicated for 5 min and diluted in the assay buffer for quantification. Electron microscopy after plastic embedding for HRP analysis 61 or after immunogold labelling of cryosections 62 has been described. Quantitation of LBPA labelling of sections from control (DMSO‐treated) or thioperamide‐treated cells was performed in a double‐blind fashion by counting the number of gold particles per endosomes in each endosome identified in two sets of 16 micrographs for each condition (DMSO‐ and thioperamide‐treated cells).

Moreau D., Vacca F., Vossio S., Scott C., Colaco A., Paz Montoya J., Ferguson C., Damme M., Moniatte M., Parton R.G., Platt F.M, & Gruenberg J. (2019). Drug‐induced increase in lysobisphosphatidic acid reduces the cholesterol overload in Niemann–Pick type C cells and mice. EMBO Reports, 20(7), e47055.

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High-Throughput Screening of Selleck Cambridge Cancer Library

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Cell Viability Assay with Presto Blue

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Example 7

Cell viability was measured using Presto Blue Cell Viability Reagent (ThermoFisher Scientific), according to the manufacturer protocol. Briefly, cells were plated overnight in complete media in 96 well plate at 1×10⁴cells per well. Cells were treated with inhibitors and/or virus for indicated time periods prior to determining cell viability. Presto Blue Cell Viability Reagent was added to cells in complete media to make a 1× final concentration and cells were incubated for 15 min at 37° C. Fluorescence was measured at EX535/EM615 using the Tecan M1000 Infinity Plate Reader (Tecan). Assay plates were then washed three times with PBS using a BioTek EL406 plate washer, and GFP fluorescence was measured at EX488/EM510 using the M1000 Infinity Plate Reader. PBS was aspirated, and cells were incubated with commercial RIPA Lysis Buffer (ThermoFisher), subjected to a freeze and thaw cycle at −20*C and 37*C respectively, and then measured again for total fluorescence. Analysis of data was done using Microsoft Excel and GraphPAD Prism for graphing and statistical analyses. These experiments were performed in triplicate 3 or more times and the average (±standard deviation) of three independent experiments was determined.

US11564919B1. Small molecule inhibitors for treatment of alpha viruses (2023-01-31). National Technology & Engineering Solutions of Sandia, LLC [US]. Inventors: Brooke Nicole Harmon [US], Oscar Negrete [US], Joseph S. Schoeniger [US], Edwin A. Saada [US].

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Immunofluorescence Staining of Cellular Organelles

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Culture medium was evacuated from 384 well imaging plates with a BioTek EL406 plate washer. Cells were fixed with cold methanol at − 20 °C for 15 min. Plates were washed once with 1xPBS before primary antibody incubation at 4 °C overnight in staining solution: 1xPBS, 1% BSA (Thermo Fisher Scientific), 0.2% Triton X-100 (Sigma-Aldrich), 5% goat serum (Jackson ImmunoResearch). Secondary staining was performed in PBS with addition of HOECHST 33,342 (Thermo Fisher Scientific). Plates were washed 3 × with PBS between staining steps. Primary antibodies were: mouse anti-p62 antibody (1:1000 dilution, MBL International, #M162-3); rabbit anti-LAMP1 antibody (1:1000, Cell Signaling Technology, #9091S). Secondary antibodies: highly cross-adsorbed secondary antibodies goat anti-mouse IgG (H + L) Alexa Fluor 568/647 and goat anti-rabbit IgG (H + L) Alexa Fluor 647 (Thermo Fisher Scientific).

Duan L., Hu M., Tamm J.A., Grinberg Y.Y., Shen F., Chai Y., Xi H., Gibilisco L., Ravikumar B., Gautam V., Karran E., Townsend M, & Talanian R.V. (2021). Arrayed CRISPR reveals genetic regulators of tau aggregation, autophagy and mitochondria in Alzheimer’s disease model. Scientific Reports, 11, 2879.

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Quantifying Antibodies against Respiratory Viruses

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Recombinant soluble trimeric forms of hRSV [24 (link)] or hMPVF [25 (link), 26 (link)] proteins were produced in 293F cells and purified by FPLC as described previously. 384-well plates (Nunc) were coated with 2 μg/mL of recombinant hRSV F or hMPV F protein and incubated overnight at 4 °C. The plate was washed 3x, incubated with blocking buffer for 1 h at room temperature, and washed once more. Serum samples were diluted serially in blocking buffer and applied to the plate in triplicate. Following overnight incubation at 4 °C, the plate was washed 4x. A 1:4,000 dilution of alkaline phosphatase-conjugated anti-monkey IgG (Fitzgerald Industries, Acton, MA) was applied to the plate and incubated for one hour at room temperature. The plate was washed 4x and then incubated with 4-nitrophenyl phosphate disodium salt hexahydrate substrate solution (Sigma) for thirty minutes. Absorbance was measured at 405 nm using a BioTek Power Wave HT plate reader. All washes were performed using a BioTek EL406 plate washer. Antigen-specific titer was determined by calculating the maximum inverse dilution that resulted in an absorbance of 0.2 greater than pre-immunization sera at the same dilution.

Bates J.T., Pickens J.A., Schuster J.E., Johnson M., Tollefson S.J., Williams J.V., Davis N.L., Johnston R.E., Schultz-Darken N., Slaughter J.C., Smith-House F, & Crowe JE J.r. (2016). Immunogenicity and efficacy of alphavirus-derived replicon vaccines for respiratory syncytial virus and human metapneumovirus in nonhuman primates. Vaccine, 34(7), 950-956.

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SARS-CoV-2 Spike Protein Binding Assay

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Wash Buffer A was made by diluting 1 L of 20X Wash Buffer A solution (Teknova, Hollister, CA) in 19 L of H₂O from a Barnstead Nanopure Water system (Thermo Scientific, Waltham, MA). After incubation with SARS-CoV-2 Spike protein, plates were washed three times with 100 μL per well of 1X Wash Buffer A. Plates were washed using a BioTek EL406 plate washer (BioTek, Winooski, VT). Blocking buffer was formulated on site by adding 10.0g bovine serum albumin (Sigma, St. Louis, MO) in 1000 mL of phosphate buffered saline (PBS, Lonza, Basel, Switzerland) with 0.05% Tween. Each well received 100 μL of blocking buffer for all plates. Plates were incubated for at least 2 hours, or wrapped in plastic wrap and incubated at 4°C overnight.

Larsen S.E., Berube B.J., Pecor T., Cross E., Brown B.P., Williams B., Johnson E., Qu P., Carter L., Wrenn S., Kepl E., Sydeman C., King N.P., Baldwin S.L, & Coler R.N. (2021). Qualification of ELISA and neutralization methodologies to measure SARS-CoV-2 humoral immunity using human clinical samples. bioRxiv.

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Immunofluorescence Quantification Workflow

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Cells were plated in 96-well glass bottom, black wall plates (GreinerOne, #655892) and grown overnight to 70% confluency. Treatments were performed as indicated. Cells were fixed for 10 min in either −20°C methanol or 4% paraformaldehyde (Electron Microscopy Sciences). Cells were blocked and permeabilized in a solution containing 1:1 Odyssey blocking buffer (LiCor)/PBS (Invitrogen) with 0.1% Triton X-100 (Sigma) and 1% normal goat serum (Invitrogen) for 1 h at room temperature. Primary antibodies were added overnight at 4°C in blocking buffer described above. After washing plates with PBS using an EL-406 plate washer (BioTek), secondary Alexa-conjugated antibodies (Life Technologies) were diluted 1:1,000 in blocking solution and applied for 1 h at room temperature. Cells were then washed again in PBS as described and imaged using an INCELL 6000 high-content imager. Images were then analyzed using HCS Explorer imaging software. Immunofluorescence quantitation was performed manually or through use of the Cell Profiler suite software (Carpenter at al., 2006 (link)).

Goodwin J.M., Dowdle W.E., DeJesus R., Wang Z., Bergman P., Kobylarz M., Lindeman A., Xavier R., McAllister G., Nyfeler B., Hoffman G, & Murphy L.O. (2017). Autophagy-independent lysosomal targeting regulated by ULK1/2-FIP200 and ATG9. Cell reports, 20(10), 2341-2356.

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Quantifying Cellular Uptake of siRNA

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A431-d2EGFP cells were seeded at a density of 15 000 cells/well in 96-well plates 16–20 h prior to the experiment. The p19-E18 constructs were incubated with fluorescently labeled siRNA (Seq I) at a 1:1 molar ratio for 30 min at 4°C. The p19-E18/siRNA complexes were then diluted in DMEM containing 10% FBS at varying concentrations and incubated with cells for 0–6 h in a reverse timecourse. For competition experiments, incubation was performed with 20 nM of p19-E18/siRNA complexes and varying concentrations (0–4 μM) of SUMO-E18 in complete media. After 6 h, cells were washed with PBS, trypsinized, neutralized with cold PBSA containing 2% FBS and analyzed on an iQue Screener (IntelliCyt). All liquid handling was performed using an EL406 plate washer (BioTek) and a Freedom EVO 150 liquid handling system (Tecan) to minimize variability. Background from untreated cells were subtracted from all measurements, which were then converted to number of fluorophores using Quantum Alexa Fluor 647 MESF beads following manufacturer's instructions (Bangs Laboratories).

Yang N.J., Kauke M.J., Sun F., Yang L.F., Maass K.F., Traxlmayr M.W., Yu Y., Xu Y., Langer R.S., Anderson D.G, & Wittrup K.D. (2017). Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins. Nucleic Acids Research, 45(13), 7602-7614.

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High-Throughput Fluorescence In Situ Hybridization

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For high-throughput FISH, cells were plated in 384-well CellCarrier plates (Perkin-Elmer) at a concentration of 80 cells/μl (~2000 cells/well). Cells were plated automatically using a Multidrop Combi (Thermo Scientific) or manually. Cells were fixed in 4% PFA in PBS for 15 minutes, permeabilized in 0.5% Saponin (Sigma Aldrich) /0.5% Triton X100/PBS for 20 minutes at RT and incubated in 0.1N HCl for 15 minutes. Cells were kept in 50% formamide/2X SSC for at least 30 minutes at room temperature. A probe mix containing 60ng of fluorescently-labelled probe, 1mg human COT1 DNA (Invitrogen), and 20μg yeast tRNA (Ambion) was ethanol precipitated, and re-suspended in 10μl of hybridization buffer (10% dextran sulphate, 50% formamide, 2X SS, 1% Tween 20). Probe mix was then manually added to each well, denatured together with cells at 85°C for 7 minutes and left to hybridize at 37°C overnight. Excess probe was washed three times with each: 1X SSC and 0.1X SSC at 42°C for 5 minutes using an automated EL406 plate washer (Biotek). Cells were finally stained with DAPI in PBS (5ng/μl) before imaging.

Shachar S., Voss T.C., Pegoraro G., Sciascia N, & Misteli T. (2015). A high-throughput imaging-based mapping platform for the systematic identification of gene positioning factors. Cell, 162(4), 911-923.

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