We performed ELISAs as previously described [15 (link)]. Briefly, spike proteins were suspended at 1 μg/ml in 1 × PBS. One hundred microliters of protein suspension was added to each well of a 96-well Nunc MaxiSorp ELISA plate and allowed to coat overnight at 4 °C for 16 h. Wells were washed three times with 300 μl of 1× PBS + 0.05% Tween20 (wash buffer) followed by blocking for 2 h at room temperature with 200 μl of 1× PBS + 0.05% Tween20 + 5% Non-fat dry milk (blocking buffer). Wells were washed again three times with 300 μl of wash buffer prior to addition of 100 μl of sample diluted in blocking buffer (serum samples were heat inactivated for 45 min at 56 °C and diluted at 1:400 in blocking buffer). Samples were incubated for 1 h at room temperature, then washed three times with 300 μl of wash buffer. One hundred microliters of 1-Step Ultra TMB Substrate (ThermoFisher) was added and the plate was incubated for 10 min prior to stopping the reaction with 1 N sulfuric acid (Stop Solution, ThermoFisher). Absorbance was read at 450 nm and 650 nm on a BioTek Epoch2 plate reader. The process is semi-automated through the use of a BioTek EL406 plate washer/dispenser and two BioStack 4 plate stackers to minimize plate-to-plate variation and increase throughput (see Klumpp-Thomas C, Kalish H et al. 2020 for detailed automation methods) [15 (link)].
El406 plate washer dispenser
Manufactured by Agilent Technologies
The EL406 plate washer/dispenser is a laboratory instrument designed for automated plate washing and liquid dispensing. It is capable of handling microplates, deep-well plates, and other common laboratory vessels. The device performs essential functions such as plate washing, liquid dispensing, and plate drying, facilitating efficient and consistent sample preparation for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
1 step ultra tmb substrate, by Thermo Fisher Scientific (2 mentions)
Epoch 2 plate reader, by Agilent Technologies (2 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (2 mentions)
Stop solution, by Thermo Fisher Scientific (2 mentions)
Fugene hd, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Fugene hd, by Promega (1 mentions)
Echo 500 550, by Beckman Coulter (1 mentions)
Pherastar fs multimode plate reader, by BMG Labtech (1 mentions)
Celltiter glo, by Promega (1 mentions)
4 protocols using el406 plate washer dispenser
1
ELISA for Spike Protein Detection
2
Automated Transfection of Vero E6 Cells
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Vero E6 cells were transfected using FuGENE HD (Promega, US) at 3.5:1 reagent to plasmid ratio. The FuGENE HD-plasmid mixes were prepared in Opti-MEM (Gibco) following the manufacturer’s protocol, after 10–15 min complex formation, a suspension of trypsinized Vero E6 cells in fully supplemented media was added, the resulting suspension was incubated at RT for 15–30 min in rotation, and the cells were seeded onto plates either manually or with automation.
In the automated assay, the transfected cells were seeded to CellCarrier Ultra 384-well microplates (PerkinElmer, US) with a seeding density of approximately 3–4 x 10ˆ3 cells/well in 25 μL per well using MultiFlo FX RAD dispenser (BioTek, US) and incubated at 37°C humidified atmosphere for 48 h in the growth media. Cells were fixed in 4% PFA for 15 min at RT, washed twice with PBS, and filled with fresh PBS. The plates were stored at 4°C prior to use. Liquid dispensing and aspiration were performed using EL406 -plate washer/dispenser (Biotek).
In the automated assay, the transfected cells were seeded to CellCarrier Ultra 384-well microplates (PerkinElmer, US) with a seeding density of approximately 3–4 x 10ˆ3 cells/well in 25 μL per well using MultiFlo FX RAD dispenser (BioTek, US) and incubated at 37°C humidified atmosphere for 48 h in the growth media. Cells were fixed in 4% PFA for 15 min at RT, washed twice with PBS, and filled with fresh PBS. The plates were stored at 4°C prior to use. Liquid dispensing and aspiration were performed using EL406 -plate washer/dispenser (Biotek).
3
ELISA Protocol for Spike Protein
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We performed ELISAs as previously described (15 (link)). Briefly, spike proteins were suspended at 1 ug/ml in 1x PBS. One hundred (100) microliters of protein suspension was added to each well of a 96-well Nunc MaxiSorp ELISA plate and allowed to coat overnight at 4°C for 16 hours. Wells were washed three times with 300 ul of 1x PBS + 0.05% Tween20 (wash buffer) followed by blocking for 2 hours at room temperature with 200 ul of 1x PBS + 0.05% Tween20 + 5% Nonfat dry milk (blocking buffer). Wells were washed again three times with 300 ul of wash buffer prior to addition of 100 ul of sample diluted in blocking buffer (serum samples were heat inactivated for 45 minutes at 56°C and diluted at 1:400 in blocking buffer). Samples were incubated for 1 hour at room temperature, then washed three times with 300 ul of wash buffer. One hundred (100) microliters of 1-Step Ultra TMB Substrate (ThermoFisher) was added and the plate was incubated for 10 minutes prior to stopping the reaction with 1N sulfuric acid (Stop Solution, ThermoFisher). Absorbance was read at 450 nm and 650 nm on a BioTek Epoch2 plate reader. The process is semi-automated through the use of a BioTek EL406 plate washer/dispenser and two BioStack 4 plate stackers to minimize plate-to-plate variation and increase throughput (see Klumpp-Thomas C, Kalish H et al. 2020 for detailed automation methods).
4
Comprehensive Chemotherapeutic Compound Screening
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A library of 627 commercially available chemotherapeutic and targeted oncology compounds were tested at 5 concentrations in 10-fold dilutions. The library consisted of 180 approved drugs, 334 investigational compounds and 113 probes (Supplementary Dataset 1 ). The chemical compounds, DMSO (negative control) and benzethonium chloride (positive controls) were added to 384-well plates using an acoustic liquid dispensing system ECHO 500/550 (Labcyte). Biobanked frozen MNCs were thawed, resuspended in CM (conditioned medium) constituted of 77.5% RPMI 1640, 10% FCS, 12.5% human HS-5 bone marrow stromal cell line derived conditioned medium and 1% penicillin and streptomycin, let recover for 3 h, and live cells counted. Compounds were first dissolved by adding 5 μL of cell free medium, followed by 20 μL cell suspension containing 5000 viable cells to each well using EL 406 plate washer-dispenser (BioTek). The plates were incubated at 37 °C in 5% CO2 for 72 h. Subsequently, CellTiter-Glo (Promega) reagent was added to all wells and cell viability was measured using a PHERA star FS multimode plate reader (BMG Labtech).
The drug responses passing the data quality assessment were included in further analysis.70 (link) Drug sensitivity scores (DSS) were calculated as shown previously.44 (link)
The drug responses passing the data quality assessment were included in further analysis.70 (link) Drug sensitivity scores (DSS) were calculated as shown previously.44 (link)
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