Plasmid pCYBB-LacZ, kindly donated by Erol Fikrig, Yale University, New Haven, CT was used to express the lacZ gene under the control of the −209 to +12 region of the CYBB promoter (Thomas et al., 2005 (link)). A mutant CYBB promoter (pmutCYBB-LacZ) was synthesized in pIDT Blue vector (Integrated DNA Technologies, Coralville, IA) to contain mutations A-mut2, A-mut3, B-mut2 and E-mut1 (Figure 2B ). The mutated promoter was amplified by PCR and cloned into the pBlueTOPO vector (Life Technologies, Coralville, IA) (Table S3 ). HL-60 cells were transfected as described above with indicated plasmids. For HDAC inhibition reporter assays, 18 h post transfection cells were treated for 6 h with 5 mM sodium butyrate (Sigma-Aldrich) and 1 µM mocetinostat. β-galactosidase activity was measured using the Galacto-Star System (Life Technologies, Coralville, IA). Luminescence was read every 2 min for 2 h. For analysis, relative light units between 54 and 64 min were averaged.
Galacto star system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Galacto-Star system is a laboratory instrument designed for the detection and quantification of galactose levels in various samples. It provides a reliable and automated method for analyzing galactose concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Luciferase assay, by Promega (2 mentions)
Fugene hd reagent, by Promega (2 mentions)
Pidt blue vector, by Integrated DNA Technologies (1 mentions)
Pbluetopo vector, by Thermo Fisher Scientific (1 mentions)
Sodium butyrate, by Merck Group (1 mentions)
Luciferase assay reagent, by Promega (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Kod plus dna polymerase, by Toyobo (1 mentions)
0.45 m filter, by Merck Group (1 mentions)
Deae dextran, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Macs cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Interleukin 2 il 2, by Roche (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Perm wash buffer, by BD (1 mentions)
21 protocols using galacto star system
1
Characterization of CYBB Promoter Activity
2
Pseudovirus Neutralization Assay Protocol
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The neutralization sensitivities of pseudoviruses were measured following a previously described protocol [35 ]. Briefly, serial dilutions of MAbs, sCD4, or fusion inhibitors in DMEM with 10 % FCS were placed in 96-well cell culture plates in triplicate. After dilution, 200 TCID50 of each respective pseudovirus suspension was added to each well. Each plate had triplicates of the respective viral control (without any inhibitor) and a cell control (without any virus). The virus-MAb mixture was incubated for 1 h (37 °C, 5 % CO2). After incubation, 104 TZM-bl cells in suspension with 30 µg/mL of DEAE-dextran were added to each well and incubated for 48 h (37 °C, 5 % CO2). Finally, the cells were washed, lysed, and the firefly luciferase activity was measured using a Galacto-star system (Life Technologies, Carlsbad, CA, USA). The percent inhibition by MAb, sCD4, or fusion inhibitor was determined by comparing the RLU in the presence and absence of an inhibitor. Each assay was repeated at least three times and validated according to the pass/fail criteria for TZM-bl cell based neutralization assay described by Sarzotti-Kelsoe et al. [36 (link)].
3
Regulation of MDR1 Gene Expression
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Luciferase reporter gene was constructed following procedure. The 5′-flanking region of human MDR1 gene (−7864 to −7817), which contains PXRE, was amplified using KOD-plus DNA polymerase (Toyobo, Osaka, Japan) in PCR and ligated into a pGL4.12 luciferase plasmid (Promega, Madison, WI, USA) that has the promoter sequence of herpes simplex virus thymidine kinase gene. LS180 cells were seeded onto 12-well plates and cultured for 16 h. The cells were then transiently transfected with the luciferase reporter plasmid (MDR1/pGL4-TK) and pCH110-β-galactosidase transfection control plasmid using the FuGENE HD Transfection Reagent (Roche Applied Science) in additive-free MEM for 24 h. The cultured medium was replaced with fresh MEM containing MK-4 or Rif and EtOH, and the cells were incubated for another 24 h. The cells were washed with PBS and lysed in Passive Lysis Buffer (Promega). Following centrifugation at 12,000× g at 4 °C for 2 min, luciferase activity in the supernatant was determined using the Luciferase Assay Reagent (Promega). A β-Galactosidase assay was performed using the Galacto-star System (Applied Biosystems). Chemiluminescence was detected using Luminescenser-MCA AB-2250 (Atto Co., Tokyo, Japan). Reporter gene activity was normalized to β-galactosidase activity.
4
Quantification of Infectious Virus Titers
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Media was recovered approximately 48 hr post-transfection and cell-free virus stocks were generated by filtering the media through 0.45 µM filters (Millipore). The TZM-bl indicator cell line was used to quantify the amount of infectious virus (Derdeyn et al., 2000 (link); Platt et al., 1998 (link); Wei et al., 2002 (link)). TZM-bl cells were seeded at 70% confluency in 24-well plates and infected by overnight incubation with virus stocks. 48 hr post infection, the cells were lysed and infectivity was measured by analyzing β-galactosidase expression using the Galacto-Star System following manufacturer’s instructions (Applied Biosystems). β-galactosidase activity was quantified as relative light units per second using a PerkinElmner Luminometer.
5
HIV-1 Pseudovirus Neutralization Assay
6
Quantifying Viral Infectivity Using TZM-bl Cells
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The TZM-bl indicator cell line was used to quantify the amount of infectious virus (72 (link)– (link)74 (link)). Briefly, cells were seeded in 24-well plates and infected by incubation with virus stocks. After 48 h postinfection, the cells were lysed and infectivity was measured by β-galactosidase expression using the Galacto-Star System following manufacturer’s instructions (Applied Biosystems). β-galactosidase activity was quantified as relative light units per second using a PerkinElmer Luminometer.
7
Quantifying HIV-1 Viral Infectivity
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4 × 106 293T cells were seeded in 10 cm plates and transfected with 10 μg of pHIV-1 and 1.25 μg of pGFP using poly(ethlyleneimine) solution (PEI) at a ratio of 5 μl PEI per 1 μg DNA. Approximately 48-h post-transfection, the media was harvested, filtered through a 0.45 μm filter and quantified using a p24Gag enzyme-linked immunosorbent assay (ELISA) (Perkin-Elmer). A total of 2.5 × 105 Jurkat cells were plated in 1 mL of medium per well in 48 well plates and infected with 25 ng of p24Gag of each virus. SupT1 cells were infected with 10 ng of p24Gag for each virus. Supernatants were first collected when syncytia were first observed in the culture infected with HIV-1NL4-3. The amount of infectious virus present at each time point was quantified by infecting the TZM-bl indicator cell line [63 (link)–65 (link)]. Infectivity was measured by the induction of β-galactosidase using the Galacto-Star™ System (Applied Biosystems).
8
CsA Washout Assay for TRIMCypA
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The CsA washout assay was previously described [9 (link)]. OMK cells endogenously expressing TRIMCypA were seeded at 1 × 105 cells per well in 24-well plates 1 day prior to infection. Cells were spinoculated (1600×g at 16 °C for 1 h, followed by 37 °C for 30 min) with GFP or LacZ VLP in the presence of 3 μM CsA (Sigma) and 5 μg/ml polybrene with or without NVP (NIH AIDS Research and Reference Reagent Program; 0.1, 1 or 10 μM in EtOH). Inoculation media was exchanged for warm media containing CsA and/or NVP as appropriate, and this was considered the zero time point. CsA was removed at the indicated times post-infection by media exchange. NVP was added to the warm media at the indicated times and removed from all reactions by media exchange. Three days after infection, the percentage of GFP-positive cells was determined by flow cytometry. For cells infected with LacZ-VLP, cells were lysed and β-galactosidase activity in cell lysates was measured using the Galacto-Star system (Applied Biosystems). Unless stated, graphs show the mean and SEM of at least 3 biological repeats.
9
Quantifying HIV-1 Viral Infectivity
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293T cells were co-transfected with pNL4-3∆Env and pVSV G. Where indicated, PR inhibitor was added. Viral supernatants were collected at 48 h post-transfection, clarified by centrifugation, and stored at −80 °C. Serially diluted viral supernatants were used to infect 1 × 104 TZM-bl cells in 96-well plates. Infections were done in triplicate with viral supernatants for each construct tested. Infectivity of the virus particles in TZM-bl cells was assessed at 48 h post-infection using a Galacto-Star System for detecting β-galactosidase activity (Applied Biosystems), as described previously [46 (link), 47 (link)].
10
HIV Transduction Efficiency Assay
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293T cells were challenged with equivalent p24 units of GFP-HIV or LacZ-HIV VLP for 72 h. For GFP-VLP, the percentage of cells expressing GFP was determined by flow cytometry using a FACS VERSE analyser (Becton–Dickinson). Cells infected with LacZ-VLP were lysed and β-galactosidase activity in cell lysates was measured using the Galacto-Star system (Applied Biosystems).
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