The thermofluor stability assay was conducted as previously described with minor changes (34 (link),35 (link)). Samples were assembled directly into a 96-well plate (USA Scientific) where each well contained assay buffer (30 mM Tris, pH 7.5; 10 mM magnesium chloride; 0.1 mM dithiothreitol), 5 μM RAD51, 25x Sypro Orange (Invitrogen), and either 0 or 2 mM ATP. Once assembled, the plate was sealed with an optical adhesive film (USA Scientific). Temperature was controlled using an AB 7500 Fast Sequence Detection System (UVM Advanced Genome Technologies Core) where the temperature ranged from 25°C to 95°C with a 1°C increment and a 1 min dwell time per cycle. Normalized fluorescence intensities were plotted versus temperature and melting temperatures (Tm) were calculated using the build-in Boltzmann equation in GraphPad (35 (link)). The data presented here are representative of three independently conducted trials with similar observations.
96 well plate
Manufactured by USA Scientific
Sourced in United States
96-well plates are a type of laboratory equipment used for various assays and experiments. They feature a grid of 96 individual wells, typically arranged in a 8 x 12 format. These plates are designed to hold small volumes of liquids, samples, or reagents, enabling parallel processing and analysis.
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Lab products found in correlation
Sypro orange, by Thermo Fisher Scientific (2 mentions)
Optical adhesive film, by USA Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Spectramax m5, by PerkinElmer (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Ha tag rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Streptavidin hrp, by PerkinElmer (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Everolimus, by Selleck Chemicals (1 mentions)
Temsirolimus, by Selleck Chemicals (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide mtt, by Merck Group (1 mentions)
3730xl sequencer, by Thermo Fisher Scientific (1 mentions)
Genemapper software, by Thermo Fisher Scientific (1 mentions)
Superscript 3 platinum sybr green one step qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
11 protocols using 96 well plate
1
Thermofluor Stability Assay of RAD51
2
Thermofluor Assay for RAD51 Stability
3
Modified Tat ELISA for Protein Detection
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Since we couldn’t find a good commercial Tat ELISA kit. We used a modified Tat ELISA protocol. Briefly, 96-well plate (USA Scientific) was coated with 0.2 μg HA-tag rabbit monoclonal antibody (Cell Signaling) per well. Wash the wells with TBST and block the wells with blocking buffer (Licor) for 1 h at room temperature. Add 100 μL sample to specific wells and incubate at 4 °C overnight. After washing with TBST, Anti-HIV1 tat antibody (Biotin) (Abcam) in blocking buffer with 1:100 dilution was added to each well and incubate at room temperature for 2 h. Wash the wells with TBST for three times. Incubate with 100 μl diluted Streptavidin-HRP (1:100 dilution) (PerkinElmer) for 30 min at room temperature. After washing with TBST for 3 times, incubate with 100 μl OPD (O-Phenylenediamine Dihydrochloride) (PerkinElmer) for 30 min at room temperature, add 100 μl stop solution (PerkinElmer) to terminate the reaction, measure the absorbance at wavelength 490 nm using SpectraMax M5.
4
Quantitative Analysis of Gene Expression
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The RNA that was purified (see “RNA extraction,” above) was also used in a qPCR analysis to determine accurately the relative expression of target genes. cDNA was generated with a FastQuant reverse transcriptase (RT) kit (Tiangen, China), according to the manufacturer’s instructions, using 1 μg RNA in a 20-μl reaction mixture at 42°C. Quantitative PCR was carried out using Fast SYBR green master mix (Thermo Fisher Scientific). Each well contained 2 ng cDNA. The final reaction volume in each well was 10 μl in 0.1-ml 96-well plates (USA Scientific, USA). The reactions were carried out on a StepOnePlus real-time PCR system (Thermo Fisher Scientific). Data were analyzed using the StepOnePlus v2.2.3 software (Applied Biosystems). All data were compared using the comparative ΔΔCT method (53 (link)). All primer pairs (see Table S1 for a complete list of primers used for all qPCR analyses) amplified their target with equal efficiencies (data not shown).
5
MTT Assay for Drug Synergism
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Cells (5 × 103/well) of quadruplet wells in 96-well plates (USA Scientific, Irvine, CA) were exposed to CIS (Selleck Chemicals, Houston, TX) or DZ-CIS for 24 h, with the final concentration of solvent DMSO (Sigma-Aldrich) never exceeding 1%. Cells were then stained with 10% 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT, Sigma-Aldrich) for 4 h and decolorized by adding 100 µl of acidic 2-propanol. The extinction of supernatant was read at an absorbance maximum of 595 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA). For drug synergism testing, human cancer cells were exposed to exponentially increasing concentrations (0 µM to 64 µM) of everolimus or temsirolimus (Selleck Chemicals, Houston, TX) plus 4 µM DZ-CIS for 24 h. The coefficient of drug interaction (CDI) was calculated based on the Combination Index Theorem by Chou T.C. and Talalay P, using a formula CDI = AB/(A + B) where CDI < 1 indicates synergism, CDI = 1 indicates additivity, and CDI > 1 indicates antagonism [26 (link)].
6
Genotyping Microsatellite Markers in Mouse Strains
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Primer pairs for microsatellite markers polymorphic between the B and X1 strains were purchased from IDT (Coralville, IA). PCR (15-μL reactions) was performed in 96-well plates (USA Scientific; Ocala, FL) using a 4-block thermocycler (BioRad, Model PTC-225 or PTC-240), as described previously [7 (link),8 (link)]. Markers with allele sizes ≥ 5% were separated with gels made of 2.5–3.5% agarose (BioExpress; Kaysville, UT). Final band sizes were discriminated by ethidium bromide staining. Microsatellite markers with allele sizes differing less than 5% were amplified using fluorescent primers synthesized at Applied Biosystems by Life Technologies (now part of ThermoFisher Scientific, Waltham, MA). Fluorescent PCR products of microsatellites markers were separated using an ABI-3730xL sequencer located at the Cincinnati Children’s Genetic Variation and Gene Discovery Core (http://dna.cchmc.org/ ), and genotypes ascertained using GeneMapper software (V4.0, ABI). For any remaining regions identified as heterozygous by the MUGA or MegaMUGA SNP panels (GeneSeek, Inc.), targeted microsatellite markers were used whenever possible to remove undesired B-alleles or to fix them in a homozygous state. For small regions with no known polymorphic microsatellite markers, individual SNPs (ABI) were used with real-time qPCR (ABI 7300).
7
Quantification of RECK Isoforms by qRT-PCR
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Cells were harvested in TRIzol reagent (Invitrogen). Chloroform was added and the lysate was centrifuged. The aqueous supernatant containing RNA was collected. Isopropanol was added to precipitate the RNA. The precipitated RNA pellet was washed with 70% ethanol, then dissolved in water and quantified by NanoDrop 2000C (Thermo Scientific). RNA (50 ng) was used for one reaction of reverse transcription and amplified using a SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen). A reaction of 25 µl was amplified in 96-well plates (USA Scientific) in triplicate for each gene. Real-time RT-PCR was performed in a CFX96 real-time system (Bio-Rad) and all results were normalized to the expression level of UBC in the same sample. Gene expression levels were calculated using the 2−∆∆Ct method. We removed outliers, which were defined as values that were ±100% of the average of the other values. Primer sequences are as follows: short RECK isoform, 5′-TTGCGCCTCTATTAGTCCACAA-3′ and 5′-AGTAATAATTGCTCATTGGCAAAA-3′; long RECK isoform 5′-CAGAAATTGTGATCCTGATCATTCC-3′ and 5′-AAAGTCGGGCTGTCAGAGTTGATA-3′; total RECK 5′-AAATGTGCCGTGATGTATGTGAAC-3′ and 5′-TTAAACACACCTGGCAAAGATGAA-3′; UBC 5′-TCTTGTTTGTGGATCGCTGTGA-3′ and 5′-CAGGAGGGATGCCTTCCTTATC-3′; CTGF 5′-CTGCAGGCTAGAGAAGCAGAGC-3′ and 5′-GCTCAAACTTGATAGGCTTGGAGA-3′.
8
Metallic Ion Dependence on Inhibition of C. difficile
9
Virus Cytotoxicity Assay in Vero E6 Cells
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Vero E6 cells were seeded into 96-well plates (USA scientific) at 1 × 104 cells per well and were cultured overnight at 37 °C. Cells were infected with a 5-fold dilution series of virus containing media in triplicate and cultured for a further 48 h. Plates were fixed in 4% paraformaldehyde (Sigma–Aldrich) for 5 min at room temperature and then stained with 0.05% crystal violet (Sigma–Aldrich) in 20% methanol (Sigma–Aldrich) for 30 min at room temperature. Plates were then washed twice in water and cell death was assessed by the presence (live cells) or absence (dead cells) of crystal violet stain. Viral load was calculated based on the dilution at which the media killed 50% of the cells using the TCID50 formula [39] .
10
Cytotoxicity Assay of Cisplatin Compounds
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Cells (1 × 104/well) of quadruplet wells in 96‐well plates (USA Scientific, Irvine, California) were exposed to exponentially increasing concentrations (0.25 μM to 64 μM) of CIS or DZ‐CIS for 24 hours. The final concentration of solvent DMSO (Sigma‐Aldrich) never exceeded 1%. Cells were treated with 10% 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (Sigma‐Aldrich) for 4 hours and decolorized with 100 μL 2‐propanol (Sigma‐Aldrich). Supernatant extinction was read at 595 nm by microplate reader (Bio‐Rad Laboratories, Hercules, California).
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