Mice were sacrificed and draining popliteal lymph nodes and tibialis anterior muscles excised. Lymph nodes were washed in PBS, crushed and cells were recovered by passage through a 70 μm strainer. Cells were lysed in NP-40 lysis buffer to release luciferase for ex vivo detection. Tibialis anterior muscles were macerated in NP-40 lysis buffer with glass beads using a Mini Beadbeater (Biospec Products) at maximum speed (4800 oscillations min−1). Homogenised material was centrifuged at 4000 rpm for 4 min, and supernatant collected. Supernatants from lymph nodes and muscle extracts were assayed for Photinus luciferase activity using the BrightGlowTM Luciferase Assay System (Promega, UK) according to manufacturers instructions. Luminescence was measured using a Varioskan Flash luminometer (Thermo Scientific).
Bright glow luciferase assay system
Manufactured by Promega
Sourced in United States
The Bright-Glow™ Luciferase Assay System is a sensitive and reliable tool for measuring luciferase reporter activity in cell-based assays. It utilizes a stable luciferase enzyme and a proprietary buffer system to generate a bright, sustained luminescent signal.
Automatically generated - may contain errors
Lab products found in correlation
Mini beadbeater, by Biospec (1 mentions)
Varioskan flash luminometer, by Thermo Fisher Scientific (1 mentions)
Synergy h4 multi mode plate reader, by Agilent Technologies (1 mentions)
Perm wash buffer, by BD (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Macsquant cytometer, by Miltenyi Biotec (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
Luminoskan ascent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Quant it picogreen dsdna reagent and kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
3 4 5 dimethylthiazole 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Trypsin, by HiMedia (1 mentions)
Prism 9, by GraphPad (1 mentions)
Flx800, by Agilent Technologies (1 mentions)
9 protocols using bright glow luciferase assay system
1
Quantifying Luciferase Activity in Lymph Nodes and Muscles
2
Quantitative Luciferase Reporter Assay
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Luciferase reporter expression was detected with the Bright glow TM Luciferase Assay System (Promega) for all in vitro studies. Cells (30,000) were grown in Labtek II, transfected with RNA alone or complexed with the delivery vehicles. 24 h post-delivery, cells were washed once with PBS, lysed with glo lysis buffer, and 100 µl of luciferin substrate reagent were added, according to the manufacturer protocol. Luminescence was measured immediately in a 96 well plate reader (Biotek Synergy H4 multimode plate reader). For hydrogel experiments, cells (0.5 X 10 6 ) were plated in a 6 well plate, transfected with IVT mRNA alone or with the delivery vehicles. 24 h post-delivery, cells were trypsinized, washed with PBS, lysed with Glo lysis buffer and the bright glow substrate reagent was added. The protein concentration of the lysates was quantified with BCA assay and the luciferase reads were normalized per mg of total protein.
3
Luciferase Assay and Flow Cytometry Protocol
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STING37shRLR cells (2 x 105 per well) were plated in a 24-well plate and infected with appropriate MOIs. For rMV2-Fluc or CHIKV-Rluc infection, at each time point cells were lysed by Passive Lysis buffer (E1941, Promega, Fitchburg, Wisconsin), and luciferase activity was measured with Bright-Glow Luciferase assay system (E2650, Promega) or Renilla Glow Luciferase Assay System (E2720, Promega), correspondingly. For immunostaining, cells were washed twice with phosphate-buffered saline (PBS) and 2% foetal calf serum (FCS) and then fixed in PBS containing 4% paraformaldehyde. Cells were permeabilized with PermWash buffer (#554723, BD Biosciences), incubated with the primary antibody at 4°C for 30 min, washed in PermWash and incubated with the secondary antibody. Cells were washed twice with PBS 2%FCS before analysis by flow cytometry using a MACSQuant cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and analysis was done with the software FlowJo (vers 7.6).
4
HIV and MLV Transduction Assay
5
PLGA-PBAE Polyplexes for Luciferase Delivery
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PLGA 50 : 50 RESOMER (RG502 H) polymer was purchased from Evonik Röhm Pharma GmbH, Germany. PBAE polymers A1 (bis(3-(propionyloxy)propyl)3,3′-(propane-1,3-diyl-bis(methylazanediyl))dipropanoate) and A2 (bis(4-(propionyloxy)butyl)3,3′-(ethane-1,2-diyl-bis(isopropylazanediyl))dipropanoate) were synthesized in our lab. Plasmid encoding firefly luciferase (pCMV-Luc) was procured from ELIM Biopharmaceuticals, USA, and propagated in large quantity in competent E. coli DH5α cells. Further the plasmid was isolated and purified using mini preparation kit from Sigma chemical Co, (St. Louis, MO, USA). Bright glow luciferase assay system was procured from Promega, USA. Quant-iT PicoGreen dsDNA Reagent and Kits were procured from Invitrogen Life Technologies Co. (USA). 3-(4,5 dimethyl thiazole-2 yl)-2,5-diphenyl tetrazolium bromide (MTT) from Sigma chemical Co, St. Louis, MO, USA was used in studies. Dimethylsulfoxide (DMSO), isopropanol were procured from Qualigens fine chemicals, Mumbai, India. Trypsin, 4-(2-hydroxyethyl)-1-Piperazineethanesulphonic acid (HEPES) sodium salt buffer saline, and normal melting agarose were procured from Himedia lab Pvt. Ltd., Mumbai, India. Lipofectamine 2000 and fetal bovine serum (FBS) were purchased from life technologies, Carlsbad, USA. All other chemicals used in the study were of analytical grade.
6
HIV Infection Quantification in T Cells
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TL3 and TC3 cells were plated at 105 cells in 500 µL of RPMI1640 culture medium in 24-well plates and infected with HIVluc viral supernatant. Cells were collected 5 days post-infection by centrifugation at 1000× g for 6 min and the pellet lysed in 100 µL of PBS-1% Tween 20 for 15 min on ice. Cellular lysates were centrifuged at 22,000× g for 2 min and supernatant was used for quantification of luciferase activity. An aliquot of 20 µL of the cellular lysate supernatant was mixed with 45 µL of substrate (Bright-Glow™ Luciferase Assay System, Promega, Fitchburg, WI, USA) and luciferase activity was quantified using a microplate luminometer.
7
HIV-1 Neutralization Assay for Monoclonal Antibodies
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The HIV-1 neutralization assays of monoclonal antibodies (mAbs) were done as described earlier.38 (link),59 (link) Neutralization was measured as a reduction in luciferase gene expression after a single round of infection of TZM-bl cells (NIH AIDS Reagent Program) with HIV-1 envelope pseudoviruses. The TCID50 of the HIV-1 pseudoviruses was calculated and 200 TCID50 of the virus was used in neutralization assays by incubating with 1:3 serially diluted mAbs starting at 10 μg/ml. After that, freshly trypsinized TZM-bl cells in growth medium (complete DMEM with 10% FBS and 1% penicillin and streptomycin antibiotics) containing 50 μg/ml DEAE Dextran and 1 mM Indinavir (in case of primary isolates) at 105 cells/well were added and plates were incubated at 37°C for 48 h. Virus controls (cells with HIV-1 virus only) and cell controls (cells without virus and antibody) were included. MuLV was used as a negative control. After the incubation of the plates for 48 h, luciferase activity was measured using the Bright-Glow Luciferase Assay System (Promega). IC50 for antibodies were calculated. Values were derived from a dose-response curve fit with a non-linear function using the GraphPad Prism 9 software (San Diego, CA).
8
Evaluating Luciferase Transfection in EL4 Cells
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The transfection ability of the pCMV-Luc microparticles on EL4 cells was evaluated using bright glow luciferase assay system (Promega, USA) in a white 96-well plate. Lipofectamine 2000 was used as standard and the relative transfection efficiency of the formulation was determined using fluorescent plate reader FLx800 from Biotek [7 (link)].
9
Neutralization Profiling of HIV-1 scFv Monoclonals
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The neutralization data of the HIV-1 positive samples (AIIMS 617, AIIMS 619, AIIMS 621 and AIIMS 627) has been discussed in detail in our previous study40 (link). The neutralization profiling of INDO-SA 2007 is recently published and that of INDO-SA 0030 was unpublished data. Briefly, different dilutions of scFv monoclonals (100 μg/ml–0.09 μg/ml) were incubated with 200 TCID of the pseudoviruses/primary isolates44 (link)45 (link)65 (link) (derived from PBMCs of HIV-1 infected Indian donors) for 1 h at 37 °C. Then TZM-bl cells were trypsinized and seeded at 104 cells/well (in DMEM, containing 25 ug/ml DEAE Dextran). In case of primary isolates, Indianavir (1 mM) was also added to the cells and plates were incubated at 37 °C. After 48 h, luciferase activity was measured (both for pseudoviruses and primary isolates) using the Bright-Glow Luciferase Assay System (Promega Inc.). IC50 values for scFv monoclonals were calculated by a dose-response curve fit with non- linear function, with the help of Graph Pad prism software (San Diego, CA). Each experiment was repeated twice and performed in duplicates and mean IC50 was calculated.
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