HCT-8 cells were seeded onto 96-well plates and infected with C. parvum transgenic oocysts expressing Nluc for in vitro growth assay as described previously (19 (link), 29 (link)). Briefly, purified transgenic Nluc-expressing oocysts (5,000 oocysts per well) were incubated with different concentrations of TMP or BKI-1294 (kind gift from Wesley Van Voorhis, University of Washington) for 48 h. No drug was added to the control wells. The culture supernatant was discarded from the wells after 48 h of incubation, and 100 μl of NanoGlo lysis buffer (Promega) was added to the wells and incubated for 15 min at 37°C and 5% CO2. The lysate was pipetted up and down several times, and 100 μl of NanoGlo lysis buffer containing 1:50 of NanoGlo substrate (Promega) was added to the wells. Lysates were transferred to white 96-well plates, and luminescence was measured on the Wallac Victor2 1420 multilabel counter (Perkin Elmer Inc.). Fifty percent effective concentration (EC50) values were calculated using a non-linear regression (curve fit of log dose versus response) in GraphPad Prism v8.
Nanoglo lysis buffer
Manufactured by Promega
Sourced in United States
The NanoGlo lysis buffer is a reagent used to prepare cell samples for analysis of reporter gene assays. It is designed to efficiently lyse cells and release cellular contents, including reporter proteins, for downstream detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Nano glo substrate, by Promega (4 mentions)
Ctg reagent, by Promega (2 mentions)
Opera qehs, by PerkinElmer (2 mentions)
Synergy h4 hybrid microplate reader, by Agilent Technologies (2 mentions)
Prism v8, by GraphPad (1 mentions)
Wallac victor2 1420 multilabel counter, by PerkinElmer (1 mentions)
Modulus microplate reader, by Promega (1 mentions)
Prism software, by GraphPad (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Nano glo, by Promega (1 mentions)
Clariostar luminometer, by BMG Labtech (1 mentions)
6 protocols using nanoglo lysis buffer
1
In Vitro Cryptosporidium Infection Assay
2
Neutralizing Antibody Assay with VLPs
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One day prior to the assay, 50,000 SW-13 cells were seeded into a 96-well black-walled, clear bottom tissue culture plate. All serum samples were heat inactivated at 56°C for 30 m. Half-log serial dilutions were made in duplicate from 1:25 to 1:25,368 and then an equal volume of medium with IbAr 10200 VLPs containing 237 TCID50 units was added and incubated at 37°C/5% CO2 for 1 h. Final effective dilutions of analyte sera ranged from 1:50 to 1:50,736. Half of this reaction mixture (50 μl) was then added to the previously aspirated target cell plate. Cells were incubated for 24 h before being lysed using NanoGlo Lysis buffer mixed with 1/50 dilution of NanoGlo substrate (Promega). Samples were mixed and incubated for 5 m at ambient temperature prior to the luminescent signal being measured on a Modulus Microplate Reader (Turner Biosystems) with an integration time of 5 s per well. To measure the effect of complement on neutralization, Low-Tox Guinea Pig Complement (Cederlane Labs) was reconstituted in DMEM, filtered, and added to the VLP/sera mixture at a final concentration of 5% and the assay was carried out as above. Data were analyzed as previously reported using GraphPad Prism software (GraphPad Software) [33 (link)].
3
Cytopathic Effect Assay for Cryptosporidium Infection
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High-content imaging HCT-8 cell infection assays with C. parvum and C. hominis were conducted following established protocols27 (link) in 384-well flat, black clear-bottom plates using an Opera QEHS (PerkinElmer) imaging system. We also developed a cytopathic effect (CPE)-based assay monitoring host cell viability using CTG reagent (Promega) that did not require staining or imaging. Further details on EC50 determination for C. parvum and C. hominis using both assays in a ten-point dose–response with threefold compound dilution will be described elsewhere (A.T.C. et al., manuscript in preparation). The EC50 of KDU731 was also determined using Nluc-expressing parasites8 (link). In brief, HCT-8 cells were infected with purified Nluc-expressing parasites (1,000 oocysts per well) and incubated with different concentrations of KDU731 for 48 h. Culture supernatant was removed from the wells and 200 μl of Nano-Glo lysis buffer containing 1:50 of Nano-Glo substrate (Promega) was added to the wells. Lysate was transferred to 96-well plates and luminescence was measured on a Synergy H4 Hybrid Microplate Reader (BioTek Instruments).
4
High Content Imaging of Cryptosporidium Infection
5
Quantitative Assays for SARS-CoV-2 VLP Binding and Entry
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For the binding assay, HeLa and HeLa-ACE2 cells were incubated with the normalized Nluc input multiplicities of EMN (E+M+N) and SEMN (S+E+M+N) containing VLPs for 1.5 h at 4 °C. The cells were washed and lysed to quantify the associated Nluc activity using the Promega’s Nano-Glo luciferase assay system. The EMN VLPs were used to calculate the background.
For the VLP-cell entry assay, HeLa cells were transfected with pcDNA-ACE2-LgBiT. Twenty-four h later, cells were inoculated with EMN and SEMN, containing VLPs for 1 h at 4 °C. Thereafter, cells were incubated with or without trypsin (20 ng/µL) in the presence of Nluc live cell substrate vivazine and transferred to 37 °C to initiate VLP-cell entry. Nluc activity was measured at various time points to assess kinetics of VLP entry. After 2 h, cells were dissolved in Nano Glo lysis buffer (Promega Corporation, Madison, WI, USA) to allow for maximal HiBiT:LgBiT complementation. The proportions of VLPs that entered cells in the 2 h time period were estimated relative to the maximal complementation after detergent cell lysis.
For the VLP-cell entry assay, HeLa cells were transfected with pcDNA-ACE2-LgBiT. Twenty-four h later, cells were inoculated with EMN and SEMN, containing VLPs for 1 h at 4 °C. Thereafter, cells were incubated with or without trypsin (20 ng/µL) in the presence of Nluc live cell substrate vivazine and transferred to 37 °C to initiate VLP-cell entry. Nluc activity was measured at various time points to assess kinetics of VLP entry. After 2 h, cells were dissolved in Nano Glo lysis buffer (Promega Corporation, Madison, WI, USA) to allow for maximal HiBiT:LgBiT complementation. The proportions of VLPs that entered cells in the 2 h time period were estimated relative to the maximal complementation after detergent cell lysis.
6
Sorbitol Synchronized Hyp1-Nluc Parasite Evaluation
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Sorbitol synchronised Hyp1-Nluc parasites were diluted into a 24-well plate at 4% HCT, 2% ring stage parasitemia with Complete Media. Compounds were added to separate wells in the following concentrations: 1x EC50 M5717 (0.56 nM), 5x EC50 M5717 (2.8 nM), 10x EC50 M5717 (5.6 nM), 5x EC50 artemisinin (40 nM), 5x EC50 chloroquine (64.5 nM), 100 µM cycloheximide and 0.05% DMSO. A blood smear was immediately taken (0 h), fixed in methanol for 30 s and stained in 10% Giemsa for 5 min. An aliquot of resuspended culture was removed at the same time and frozen in a 96-well plate at -20°C. Parasites were returned to 37°C. Over the following 72 h, another blood smear and aliquot were taken at each 24 h time point. Results were determined by counting 1000 cells for each treatment and time point, separating parasites based on their appearance into either specific blood-stage or abnormal and/or dead. Images were taken with a Leica IC50 HD camera attached to a Leica DM750 microscope. Nanoluciferase (Nluc) bioluminescence was measured from the Hyp1-Nluc parasites by lysing the parasite culture 1:5 with 1x Nano-Glo Lysis Buffer (Promega, USA), and the addition of 1:1000 Nano-Glo (Promega). The bioluminescent signal was measured using a CLARIOstar luminometer (BMG Labtech) at 3000 gain for 1 s/well.
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