For titration ELISA, purified IgGs were titrated from 3.18 μg/mL- 0.001 ng/mL on 30 ng/well of the following antigens: S1-S2-His (High Five cell produced), RBD-mFc (High Five cell produced), S1-mFc (High Five cell produced) and TUN219-2C1-mFc (as control for unspecific Fc binding). In addition, all scFv-hFc were also tested only at the highest concentration (3.18 μg/mL) for unspecific cross-reactivity on Expi293F cell lysate (104 cells/well), BSA (1% w/v) and lysozyme. IgGs were detected using goat-anti-hIgG(Fc)-HRP (1:70000, A0170, Sigma). Titration assays were performed using 384 well or 96 well microtiter plates (Greiner Bio-One) using Precision XS microplate sample processor (BioTek), EL406 washer dispenser (BioTek) and BioStack Microplate stacker (BioTek). EC50 were calculated with by GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve. Titration ELISAs on other coronaviruses and S1-HIS mutants were performed as described above.
Biostack microplate stacker
Manufactured by Agilent Technologies
Sourced in United States
The BioStack Microplate Stacker is a compact, automated microplate handling system designed for use in life science laboratories. It can hold up to 50 microplates and efficiently transport them between instruments, providing a hands-free solution for high-throughput workflows.
Automatically generated - may contain errors
Lab products found in correlation
El406 washer dispenser, by Agilent Technologies (2 mentions)
Goat anti higg fc hrp, by Merck Group (2 mentions)
Precision xs microplate sample processor, by Agilent Technologies (2 mentions)
Rpmi 1640 l glutamine, by Thermo Fisher Scientific (1 mentions)
Recombinant human gm csf, by R&D Systems (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Isotype control abs, by R&D Systems (1 mentions)
Epics xl, by Beckman Coulter (1 mentions)
Prism 9, by GraphPad (1 mentions)
384 well microtiter plate, by Greiner (1 mentions)
6 protocols using biostack microplate stacker
1
SARS-CoV-2 Antibody Titration ELISA
2
Regulation of Lipid Metabolism by miR-1322
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HepG2 cells in 6‐well plates were transfected with hsa‐miR‐1322 (100 nmol/L), miRNA mimic negative control (100 nmol/L), hsa‐miR‐1322 inhibitor (200 nmol/L) or miRNA inhibitor control (200 nm). After transfection for 24 hours, cell lysates were extracted as described previously.20 Triglyceride and total cholesterol levels were determined using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, China) normalized to protein concentration following the manufacturer's instructions. The assay sensitivity was 0.01 mmol/mL, and average intra‐ and inter‐assay coefficients of variation were 3% and 5%, respectively. The absorbance was measured using a BioStack Microplate Stacker (BioTek, WI, USA) according to the instruction manual.
3
Recombinant cytokine-mediated dendritic cell differentiation
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Recombinant human GM-CSF and IL-4 were purchased from R&D Systems (Minneapolis, MN, USA). PE-labeled antibodies (Abs) against human CD80, CD86 and MHC II were from BD Pharmingen (San Diego, CA, USA) and isotype control Abs from R&D Systems (Minneapolis, MN, USA). Complete Freund's Adjuvant, InComplete Freund's Adjuvant and Lipopolysaccharide (LPS; Escherichia coli, O111:B4) were obtained from Sigma (St Louis, MO, USA). The goat anti-mouse HRP-conjugated secondary ab (goat anti-mouse IgG-HRP SC-2005, Lot # D1614) was purchased from Santa Cruz Biotechnology (San Diego, CA, USA). The BioFXr TMB super-sensitive one-component HRP microwell substrate was from SurModics (Minneapolis, MN, USA) and the BioStack Microplate Stacker from BioTek Instruments (Winooski, VT, USA). RPMI-1640 (-L glutamine) and L glutamine were from Thermo Fisher (Waltham, MA, USA) and heat-inactivated fetal bovine serum (FBS) from Sigma (F4135-500, 15H095H1).
4
Antiviral Efficiency of 6-TG Against HSV-1
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HCECs were treated with different concentrations of 6-TG for 2 h before HSV-1 infection. After HSV-1 infection and then being washed with DMEM, HCECs were treated with 6-TG again. HSV-1 infection was measured by In-Cell Western blot assay, and viral titers were titrated by PFU assay at 24 h postinfection after three freeze-thaw cycles. The 50% effective concentration (IC50) of 6-TG was calculated by linear regression of the viral inhibition curves. Viral inhibition (%) was calculated as follows: viral inhibition (%) = [1 − (number of plaques) 6-TG/(number of plaques) control] × 100.
After infection with HSV-1/green fluorescent protein (GFP), Vero cells in 96 wells were incubated with serial dilution of 6-TG, and the plates were scanned using BioStack microplate stacker (BioTek Instruments, Winooski, VT, USA) at 24 h postinfection. Viral infection (%) was calculated as follows: viral infection (%) = (fluorescence6-TG – fluorescencecell control)/(fluorescencevirus − fluorescencecell control) × 100.
After infection with HSV-1/green fluorescent protein (GFP), Vero cells in 96 wells were incubated with serial dilution of 6-TG, and the plates were scanned using BioStack microplate stacker (BioTek Instruments, Winooski, VT, USA) at 24 h postinfection. Viral infection (%) was calculated as follows: viral infection (%) = (fluorescence6-TG – fluorescencecell control)/(fluorescencevirus − fluorescencecell control) × 100.
5
Baicalein Inhibits HSV-1 Infection
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After infection with HSV-1-EGFP, the cells in 96 wells were incubated with serial dilution of baicalein, and the plates were scanned using BioStack Microplate Stacker (BioTek Instruments, Winooski, VT, USA) at 24 h post-infection. Viral infection (%) was calculated using Eq. (2) :
Viral plaque inhibition (%) was calculated as described in Section2.4 . For fluorescence microscopy and flow cytometry analysis, the infected HaCat cells were treated with baicalein as describe above, and then detected under fluorescence microscope (IX51, Olympus Co., Tokyo, Japan) or measured by flow cytometry (Epics XL, Beckman Coulter, CA, USA) at 24 and 48 h post-infection.
Viral plaque inhibition (%) was calculated as described in Section
6
Quantifying SARS-CoV-2 Antibody Responses
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For titration ELISA, sera were diluted 1:100 to 1: 9.19 × 107 in 384-well microtiter plates (Greiner Bio-One) coated with 30 ng/well of the respective RBD variant. In addition, all sera were also tested at the lowest dilution (1:100) for determination of unspecific cross-reactivity on Expi293F cell lysate (30 ng/well), BSA (30 ng/well), and lysozyme (30 ng/well). IgGs in the sera were detected using goat-anti-hIgG(Fc)-HRP (1:70,000, A0170, Sigma). Three-hundred-eighty-four-well liquid handling was performed with a Precision XS microplate sample processor (BioTek), EL406 washer dispenser (BioTek), and BioStack Microplate stacker (BioTek). OD450 nm-620 nm was measured in an ELISA plate reader (BioTek Instruments, Epoch) and its software Gen5 version 3.03 was used to calculate EC50 values, further expressed as relative potency towards an internal calibrant for which the Binding Antibody Unit (BAU) was calculated using the WHO International Standard 20/136 in relation to the original Wuhan strain RBD. The graphics were created by GraphPad Prism 9.1. Significance was calculated by pairwise non-parametric multiple comparison ANOVA (Friedman’s test) with Dunn’s multiple comparisons test, using the Wuhan wt RBD values as the reference value for all three VOCs, but Omicron data were shown separately for better illustration.
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