Host cell and intracellular T. cruzi amastigote numbers were assessed as described with minor modifications [34 (link)]. Briefly, NHDF were plated at 1.5 x 103 per well in 384 well plates and infected with MOI 1.25 for 2 hours before incubation in phenol-free media at the indicated glucose concentration. At 18 hpi, the indicated concentration of 2-DG was added, and at 66 hpi media was removed and host cell number and parasite number were assessed using 10 μL of CellTiter-Fluor (Promega) and 10 μL of Beta-Glo (Promega) per well, respectively.
Beta glo
Manufactured by Promega
Sourced in United Kingdom
Beta-Glo is a luminescent assay system used for the quantitative measurement of beta-galactosidase activity. It utilizes a substrate that emits light upon hydrolysis by beta-galactosidase, enabling the detection and quantification of this enzyme in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter fluor, by Promega (2 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
384 well plate, by Corning (2 mentions)
Bright glo, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Af2200, by Eppendorf (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Dmem f12, by Corning (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Magnesium chloride hexahydrate, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Gibco glutamax, by Thermo Fisher Scientific (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Edta free halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
13 protocols using beta glo
1
Quantifying Host-Parasite Interactions
2
Infection Assay for Trypanosoma cruzi
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Multiplex assays were modified slightly from previous work (14 (link)). At 1 day prior to infection, HFFs were trypsinized and seeded in 384-well plates (Corning) at a density of 1,500 cells per well in 30 µl DMEM-10. For infection, 10 µl of purified trypomastigotes per well was added at a multiplicity of infection of 1.25. At 2 h postinfection (hpi), plates were washed twice with PBS and replaced with 30 µl fresh DMEM-2 without phenol red. At 66 hpi, medium was removed and 10 µl CellTiter-Fluor (Promega) was added per well. Fluorescence was measured using an EnVision plate reader (PerkinElmer). Subsequently, 10 µl Beta-Glo (Promega) was added per well and luminescence was measured using an EnVision plate reader (PerkinElmer) following a 30-min incubation at room temperature. Background luminescence values from uninfected wells were subtracted from the values determined for infected wells to account for luminescence specific to host cells.
3
Measuring Tula-βgal Parasite Load
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Tula-βgal parasite load was measured using luminescence as described previously (Caradonna et al., 2013 (link); Shah-Simpson et al., 2017 (link)). One day prior to infection NHDFs were seeded in 384-well plates (Corning, Corning, New York) at a density of 1,500 cells per well and allowed to attach. Purified trypomastigotes were added at a multiplicity of infection (MOI) of 1.25 and allowed to invade for 2 hr, followed by two washes with PBS and subsequent addition of DMEM-2 without phenol red. Treatments were initiated at 18 hr post infection (hpi) to avoid any potential impacts of trypomastigote invasion and/or differentiation. At the indicated time points, growth media was removed and 10 μl Beta-Glo (Promega, Madison, Wisconsin) was added per well. Plates were incubated for >30 min at room-temperature to allow the reaction to reach equilibrium and read using an EnVision plate reader (PerkinElmer, Waltham, Massachusetts). Luminescence from uninfected wells was determined for each treatment and subtracted from infected wells to account for signal not derived from parasites.
4
Electroporation and Reporter Assays in GH4 Cells
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Electroporation experiments and reporter assays were performed as described [44 ]. GH4 cells were harvested with an EDTA solution, and 6 x 106 cells were used for each electroporation. Trypan Blue exclusion before electroporation ranged from 95% to 99%. The voltage of the electroporation was 1550 volts. This resulted in Trypan Blue exclusion of 70% to 80% after electroporation. The transformed cells were plated in 96 well dishes (Falcon Plastics) at 1 x 105 cells/well in DMEM with 10% hormone-depleted serum. The cells were allowed to attach and hormones were added for 24 h. The medium was replaced with assay buffer and the plates were frozen at -80°C. Luc assays were performed using reagents and protocols from Promega.
Control of transfection efficiency was performed using an RSV-β-galactosidase. β-galactosidase activity in the cell lysates was determined using Beta Glo (Promega, Madison, WI). Transfection efficiency did not vary significantly among transfections performed at the same time. The relative light units (RLU) of Luc activity was then corrected for minor variations in β-Galactosidase activity by converting the RLU to RLU / β-Galactosidase activity/mg protein. The fold stimulation or inhibition was then determined.
Control of transfection efficiency was performed using an RSV-β-galactosidase. β-galactosidase activity in the cell lysates was determined using Beta Glo (Promega, Madison, WI). Transfection efficiency did not vary significantly among transfections performed at the same time. The relative light units (RLU) of Luc activity was then corrected for minor variations in β-Galactosidase activity by converting the RLU to RLU / β-Galactosidase activity/mg protein. The fold stimulation or inhibition was then determined.
5
SARB Hybrid Wnt Signaling Assay
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HCT116 cells (20 × 103) were co-transfected for 6 h before 24 h treatment with 10, 20, 50 µM SARB hybrid either with 100 ng Super8xTOP-FLASH or 100 ng Super8xFOP-FLASH, respectively, and 10 ng ß-galactosidase reporter plasmid for normalization of protein amounts. Plasmids were kindly provided by Prof. Trevor Dale, Cardiff University School of Biosciences. The reporter activity was assessed by adding 50 µl of Beta-Glo (#E4720, Promega) or 50 µl of Bright-Glo (#E2610, Promega) to the 50 µl of lysed samples, following the manufacturer instructions and measured by a luminometer (VictorTM X3 Multilabel reader). The results were normalized to total protein amounts and were expressed relative to DMSO controls.
6
Yeast-based Protein-Protein Interaction Assay
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The GAL4 DNA binding domain (BD) of pGBKT7 (Clontech) was fused to the N-terminus of full-length BES1 or BES1-D protein, and the GAL4 activation domain (AD) of pGADT7 (Clontech) was fused to the N-terminus of BAF1 protein. Two constructs were co-transformed into yeast strain Y187 using the LiAc method and screened using SD-Trp–Leu medium. The optical density (OD) was read as absorbance at 600 nm using a multi-mode plate reader (Eppendorf AF2200). β-Galactosidase activity was assessed using a commercially available luminescent β-galactosidase substrate Beta-Glo (Promega, E4740), which is cleaved to release d -luciferin as a firefly luciferase substrate (de Almeida et al., 2008 (link)). The LUC values were normalized to the culture absorbance OD600.
7
Dimerization Assay for IL2RB/IL2RG
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Example 11
PathHunter U2OS IL2RB/IL2RG Dimerization Assay
U2OS IL2RB/IL2RG (DiscoverX, 93-0998C3) cells were thawed and cultured in complete DMEM/F-12 (Corning) with 100 IU Penicillin/100 μg/mL Streptomycin (Corning), 2 mM GlutaMax (Gibco), 10% heat-inactivated fetal bovine serum (FBS) (Sigma). Additional 250 μg/mL hygromycin, and 500 μg/mL G418 were added for selection. One day before the aldesleukin or variant treatment, 25 μL of cells were seeded at 0.075×106 cells/mL for a total of 1,875 cells per well in complete culture medium in a standard white TC-coated 384-well plate. On assay day, cells were treated with 25 μL of serial dilution of aldesleukin or variant samples (1:8 serial dilution of 1 μM starting concentration). Cells were incubated at 37° C., 5% CO2 for 24 hours. 30 μL of reconstituted Beta-Glo (Promega) reagent was added and allowed to incubate for 25 minutes at room temperature with shaking. Plates were read on the Envision plate reader (PerkinElmer) and luminescence readings were converted to % relative signal using the 1 μM aldesleukin treated cells as controls. Data was fitted with non-linear regression analysis, using log (against) vs. response, variable slope, 4-parameter fit equation using GraphPad Prism. Data was expressed as % relative signal vs. dose of aldesleukin or variant samples in nM.
8
Cell Proliferation Assay Protocol
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CellTiter 90® AQ one solution cell proliferation assay, pSV-40 β-Galactosidase control vector, Beta-Glo® and pGL4.13[luc2/SV40] vector, were supplied by Promega (Southampton, UK). Ninety-six well Corning cell culture plates, hydrochloric acid (purity ≥ 98%) were supplied by VWR (Fontenay-sous-Bois, France) and Luciferin was supplied by Perkin Elmer (Waltham, MA, USA). T-PER™ tissue protein extraction reagent, EDTA free halt protease inhibitor cocktail, Lipofectamine 2000 and acetonitrile HPLC grade (purity ≥ 99.5%) were supplied by Thermo Scientific (Loughborough, UK). Visking dialysis tubing was supplied by Medicell Membranes Ltd. (London, UK). Glycol chitosan Mw = 113 kDa, Mn = 98 kDa was supplied by Wako (Osaka, Japan). Deuterium oxide, sodium acetate anhydrous (purity ≥ 99%), glacial acetic acid, hyaluronidase Type I S-400–1000 units mg−1, trifluoracetic acid (purity ≥ 99%), hyaluronic acid (Mw 70–90 kDa), bovine serum albumin, Triton™ X-100, magnesium chloride hexahydrate (purity ≥ 96%), trypsin (0.02% EDTA, Gibco™ GlutaMAX, penicillin/streptomycin, minimal essential medium eagle (MEME), OptiMEM and foetal bovine serum were all supplied by Sigma Aldrich (Gillingham, UK).
9
HIV Inhibition Assay with Lectins
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HIV inhibition was measured using a luminescence-based β-galactosidase assay (Beta Glo, Promega) with TZM-bl target cells [54 ]. Serial dilutions of lectins were added to the virus at 200 TCID50 in half-area 96-well plates (Costar) and incubated for designated time periods at 37°C. TZM-bl cells were then added along with DEAE-Dextran (10 μg/ml; Sigma) and incubated for 48 h. Each condition was tested in duplicate or triplicate. Assay controls included replicate wells of TZM.bl cells alone (cell control) and TZM-bl cells with virus alone (virus control). Percentage of inhibition was determined on the basis of virus control under the specific assay conditions. Virus input corresponded to titrated stocks yielding 200 TCID50 in RLUs.
10
Quantitative RT-PCR and Immunoblot Analysis of NRF2 and FN3K Pathways
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Quantitative RT-PCR (qPCR) and immunoblots were performed as before [14 (link)]. We used the following taqman probes for qPCR analysis: HUMAN—NRF2 (Hs00975961_g1, Life Technologies, Carlsbad, CA, USA), FN3K (Life Technologies, Hs_00223368_m1), GUSB (Life Technologies, 4333767 F), and ACTB (Life Technologies, 4332645); MOUSE—Nqo1 (Life Technologies, Mm01253561_m1), Nrf2 (Life Technologies, Mm00477784_m1), Fn3k (Life Technologies, Mm00445584_m1), cMyc (Life Technologies, Mm00487804_m1) and Actb (Life Technologies, Mm00607939_s1). For immunoblotting, we used the following antibodies from Cell Signaling (Danvers, MA, USA): NQO1 (CST, 62262), MYC (CST, 5605), BCL2 (CST, 3498), KEAP1, (CST, 8047), CUL3 (CST, 2759), GAPDH (CST, 5174), and β-actin (Sigma-Aldrich A5441 St. Louis, MO, USA). Proteins were visualized using LI-COR Lincoln, NE, USA detection system after incubation with following secondary antibodies: Goat anti-rabbit-IR800 (LI-COR, 926–32211) and Goat anti-mouse-IR680. We used the following reporter assays as per manufacturer’s instructions: Luciferase-Glo (Promega) and Beta-glo (Promega). T7 endonuclease assays were conducted to confirm gene editing (NEB, Ipswich, MA, USA).
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