MMR assay was performed as described, except H3-labeled dATP and dTTP substituted for dATP and dTTP. At multiple time points, aliquots of the reaction were spotted on DEAE Filtermat (Perkin Elmer 1450-522) and dried thoroughly. Next, the DEAE Filtermat was washed 3× in 5% NaH2PO4 2× in MilliQ water, and dried overnight. The next day, MeltiLex solid scintillator (Perkin Elmer 1450=441) was added to the Filtermat at 80°C and it was incubated at room temperature. The amount of H3 isotope per spot was estimated by scintillation counting in a MicroBeta2 Plate Counter (Perkin Elmer 2450-0010) for 5 min.
Filtermat
Manufactured by PerkinElmer
Sourced in United States
The Filtermat is a versatile laboratory equipment designed for efficient sample filtration. It features a durable, corrosion-resistant construction and a user-friendly interface. The Filtermat's core function is to facilitate the separation of solid particles or suspended matter from liquid samples, enabling accurate analysis and sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Meltilex a, by PerkinElmer (2 mentions)
3h uracil, by Biotrend (2 mentions)
Microbeta2 plate counter, by PerkinElmer (1 mentions)
Deae filtermat, by PerkinElmer (1 mentions)
Betaplate scint solution, by PerkinElmer (1 mentions)
Microbeta trilux, by PerkinElmer (1 mentions)
Filtermat b, by PerkinElmer (1 mentions)
0.45 m membrane, by Merck Group (1 mentions)
Branched polyethylenimine, by Merck Group (1 mentions)
Wallac microbeta plate reader, by PerkinElmer (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
96 well flat bottom plate, by Thermo Fisher Scientific (1 mentions)
5 protocols using filtermat
1
Radioactive Assay for DNA Synthesis
2
Measuring Mycobacterial Metabolic Activity
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Extracellular bacteria (2 × 106 cells/mL) were distributed into 96-well flat bottom plates (Nunc; ThermoFisher, Bremen, Germany) in triplicates and incubated for 3 days at 37 °C/5% CO2. Next, 3H-Uracil (0.3 µCi/mL, Biotrend, Köln, Germany) was added overnight at 37 °C/5% CO2. Mtb were then fixed and killed with 4% paraformaldehyde (PFA) at room temperature (RT) for 20 min, and were then harvested (Cell harvester; Inotech, Derwood, MD, USA) onto a filtermat (Perkin Elmer, Waltham, MA, USA). Afterwards, wax plates (Meltilex A; Perkin Elmer) containing scintillation liquid were molten onto the mats. Samples were measured with a beta counter (Hidex sense micro beta counter; Turku, Finland) and the mean values of the triplicates were calculated.
3
Cell Proliferation and Senescence Assays
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For 3H-Thymidine proliferation assay, the cells were treated as described before and 1 d after reseeding in 96 well plates, the adherent cells were pulsed for 12 hours with 3H Thymidine solution (Hartmann Analytic). After incubation, the cells were transferred to a membrane Filtermat (Perkin Elmer). The membrane was washed, developed with Betaplate Scint solution (Perkin Elmer) and measured in a Luminescence Counter (MicroBeta TriLux, Perkin Elmer).
SA β-Gal staining was performed on melanoma cells reseeded for 1 d after CM exposure with the SA β-Gal staining kit (United States Biological) according to the manufacturer’s instructions.
SA β-Gal staining was performed on melanoma cells reseeded for 1 d after CM exposure with the SA β-Gal staining kit (United States Biological) according to the manufacturer’s instructions.
4
HIV-1 Virus Quantification and Infectivity Assay
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The HEK293T and HeLa cell lines were transfected with HIV-1 proviral DNA using Lipofectamine 2000 (Invitrogen). At 48 h posttransfection, virus-containing supernatants were filtered through a 0.45-µm membrane (Merck Millipore). The amount of virus in the supernatants was quantified by RT assay. RT assays were performed as described previously (126 (link)) with minor modification. Briefly, after incubation of the virus supernatants with RT reaction mixtures, which contained a template primer of poly(rA) (5 µg/ml) and oligo(dT)12-18 primers (1.57 µg/ml), in 50 mM Tris (pH 7.8), 75 mM KCl, 2 mM dithiothreitol, 5 mM MgCl2, 0.05% Nonidet P-40, and 0.25 µCi of 32P-dTTP at 37°C for 3 h, the mixtures were spotted onto Filtermat B (Perkin Elmer; catalog number [no.] 1450-521) soaked in 0.5% (vol/vol) branched-polyethylenimine (Merck Millipore; catalog no. 402727). After washing the spotted Filtermat with 2× SSC buffer (300 mM NaCl and 30 mM sodium citrate), levels of bound 32P were measured on a Wallac MicroBeta plate reader (PerkinElmer). The 50% tissue culture infective dose (TCID50) of the virus stocks was determined using TZM-bl cells.
5
Mycobacterium tuberculosis Growth Kinetics Assay
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Extracellular Mycobacterium tuberculosis bacilli (2 × 106) were distributed into 96-well flat bottom plates (Nunc) in triplicate and incubated for 3 days at 37 °C/5% CO2. Next, 3H-Uracil (0.3 µCi/mL, Biotrend Chemikalien GmbH, Köln, Germany) was added overnight at 37 °C/5% CO2. The bacteria were then fixed and killed with 4% paraformaldehyde (PFA) at room temperature (RT) for 20 min and then harvested (Cell harvester; Inotech Bioscience LLT, Derwod, MD, USA) onto a filtermat (Perkin Elmer, Waltham, MA, USA). Afterward, wax plates (Meltilex A; Perkin Elmer) containing scintillation liquid were molten onto the mats. The samples were measured with a beta counter (Hidex sense micro beta counter), and the mean values of the triplicates were calculated [30 (link)].
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