SEVI was generated as described (8 (link)). To analyze the activity of candidate microbicides in the presence of SEVI, 5 × 103 TZM-bl cells were seeded into 96-well flat-bottom plates (100 μl). The next day, when cells were ~70 % confluent, the medium was replaced by fresh medium (160 μl) in the presence of antivirals (20 μl). After 1 hour incubation, SEVI was mixed 1:1 (volume/volume) with virus in order to obtain SEVI concentrations of 35 μg/ml during virion treatment. After incubating the SEVI/virus mixture for 5 min, 20 μl of the SEVI-virus (or PBS-virus control) were then added to compound-treated cells (volume 180 μl). 3 days later, infection rates were measured using the Gal-Screen system (Applied Biosystems). The effect of SEVI on the antiviral activity of the protease inhibitor was analyzed in CEMx-M7 cells essentially as described above for the analysis of semen, except that virions were treated with 35 μg/ml of SEVI instead of semen.
Gal screen system
Manufactured by Thermo Fisher Scientific
The Gal-Screen system is a laboratory instrument designed for cell-based assays. It is used to measure and analyze cellular responses, such as gene expression or cell viability, in a high-throughput manner. The system utilizes a sensitive luminescence-based detection method to quantify the activity of reporter genes or other cellular readouts.
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12 protocols using gal screen system
1
Evaluating Antivirals with SEVI-Mediated HIV
2
TZM-bl Neutralization Assay for Virus Neutralization
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The TZM-bl neutralization assay was used as previously described [27 (link)]. The IC50, or reciprocal plasma dilution at which 50% of the virus was neutralized, for each plasma-virus pair was calculated using linear interpolation from the neutralization curve. A plasma sample was considered to be below the detectable limit of neutralization if the starting dilution (1:100) did not show >50% neutralization. For each virus plasma pair, two independent experiments were performed in duplicate unless indicated and a log2 average was calculated. For the majority of experiments, any plasma-virus pairs that demonstrated >2-fold difference in IC50 across two experiments was repeated in a third experiment and a final log2 average was calculated. Neutralization assays performed for the breadth and potency comparison including the new 9 SI cases and 27 matched controls was performed on a Tecan Freedom Evo 150 liquid automated system at a starting dilution of 1:100 and final dilution of 1:1600. β–Galactosidase activity for these TZM-bl neutralization assays was determined using the Gal-Screen System (Applied Biosystems).
3
Quantifying Yeast LacZ Expression
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Strains expressing the LacZ gene were evaluated for beta-galactosidase activity through the chemiluminescent Gal-Screen system (Applied Biosystems). Yeast cultures were grown for 16 hours to mid-log phase from a starting OD600=0.005. Prior to the assay, cultures were diluted with fresh media to approximately OD600=0.01 to 0.07. OD600 was measured, and then cultures were treated with Gal-Screen Reaction Buffer according to the manufacturer's instructions. Luminescence was quantified using a Mithras LB 940 luminometer (Berthold Technologies). Day to day variation was avoided by measuring all samples on the same day. The average luminescence across biological replicates was calculated.
4
Inhibition of CXCR4-tropic HIV Infection
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Viral stocks of CXCR4-tropic NL4-3 were generated by transient transfection of HEK293T cells with proviral DNA as described before33 (link). Inhibition of viral infection was performed in TZM-bl reporter cells. For this 70 μL of 1 × 105 cells (in growth medium supplemented with 2.5% FCS) were pretreated with 10 μL of inhibitors for 15 min at 37 °C. Cells were then inoculated with 20 μL of diluted virus. Infection rates were determined after 3 days using Gal-Screen system (Applied Biosystems).
5
Quantifying Yeast LacZ Expression
6
Yeast Galactosidase Activity Assay
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Colonies on selection synthetic defined medium by the yeast spotting assay were inoculated into 5 mL YPD medium and then incubated at 30 °C on a shaker at 300 rpm. The solution was diluted to yield an A600 of 0.05 (5 × 105 cells·mL−1) with sterilized water. Then 50 μL of cells was added to the mixed solution, which was made of 2 μL Gal‐Screen buffer A and 48 μL Gal‐Screen buffer B in Gal‐Screen System (Applied Biosystems). After incubation of the mixture at 30 °C for 1 h, 75 μL of the mixture was placed in a microplate in a Centro XS3 LB960 (Berthold Technologies), and the luminescence was measured for 0.1 s·well−1. The amount of β‐galactosidase corresponding to cleavage enzyme activity was calculated using a standard curve, which was generated with β‐galactosidase (TOYOBO). The results were expressed as the mean ± standard error. Statistical analysis was performed with JMP software (Ver. 12). A statistical difference was determined using a two‐sided Welch’s t‐test, and a difference with P < 0.05 was considered significant.
7
Inhibition of HIV-1 Infection by SP
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To examine the inhibition effect of human SP on HIV-1 infection, 1 × 105 TZM-bl cells/ml were seeded into 96-well flat-bottom plates (100 μl). The next day, the medium of the cells was replaced by X-VIVO 15 chemically defined medium (Lonza) supplemented with 0.05 mM aminoguanidine (41 (link)) and gentamicin (50 μg/ml). SP diluted in PBS was added to cells and incubated for 1 hour at 37°C. After incubation, cells were infected with X4-tropic or R5-tropic HIV-1 by spinoculation (30 min, 2090g, 37°C) and incubated for another 2 hours. Final inoculum was discarded, cells were washed with PBS, and 200 μl of X-VIVO 15 medium [+ 0.05 mM aminoguanidine, gentamicin (50 μg/ml)] was added. Infection rates were measured 2 days after infection using a Gal-Screen system (Applied Biosystems).
8
Semen Inactivation of Viral Infectivity
9
Antiviral Efficacy of Semen on HIV-1
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Five thousand TZM-bl cells were seeded in 100 µL supplemented DMEM in 96-flat-well plates. The next day, the medium was replaced by 280 µL of supplemented DMEM +50 µg/mL gentamycin. Semen samples were thawed, briefly vortexed, and diluted in PBS. Semen or PBS was mixed 1:1 with 2-fold serial dilutions of HIV-1 T/F IMCs to obtain semen concentrations of 50%, 10%, 2%, and 0.4% during virus incubation. After 5 min incubation at room temperature (RT), TZM-bl cells were infected with 20 µL of virus:semen mixture. At 2 hpi, inocula were removed, cells were washed with PBS, and 200 µL of supplemented DMEM +50 µg/mL gentamycin was added. At 48 hpi, infection rates were measured by assessing β-galactosidase activity in cell lysates using the Gal-Screen system (Applied Biosystems) supplemented with 0.16% Triton-X (Sigma #T9284). β-galactosidase-induced luminescence due to substrate conversion was quantified using an Orion microplate luminometer (Berthold).
10
Polyamines Modulate HIV-1 Infection in TZM-bl Cells
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To examine the influence of polyamines on X4-tropic and R5-tropic HIV-1 infection, 1 × 105 TZM-bl cells/ml were seeded into 96-well flat-bottom plates (100 μl). The next day, the medium was changed to 170 μl of X-VIVO 15 chemically defined medium (Lonza) and 20 μl of substances was added. TZM-bl cells were incubated for 1 hour at 37°C before they were inoculated with 10 μl of virus (cell treatment). Alternatively, the virus was treated for 15 min with the polyamines before 20 μl of the mixture was added to TZM-bl cells in 180 μl of X-VIVO 15 medium, thereby diluting the mixture 10-fold. To analyze SP-derived library fractions, TZM-bl cells in 85 μl of X-VIVO 15 medium were preincubated with 5 μl of the respective fraction. Cells were inoculated with 10 μl of virus after 30 min. Infection rates were measured 2 days after infection using a Gal-Screen system (Applied Biosystems).
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