Tritiated thymidine

Manufactured by PerkinElmer

Sourced in United States

Tritiated thymidine is a radiolabeled nucleoside used in various research applications. It is a synthetic form of the DNA base thymidine, with tritium atoms incorporated into its structure. Tritiated thymidine can be used to study cell proliferation and DNA synthesis.

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Lab products found in correlation

Microbeta trilux, by PerkinElmer (2 mentions) Annexin 5 fitc, by BD (2 mentions) A549 cell, by American Type Culture Collection (2 mentions) Propidium iodide, by Thermo Fisher Scientific (2 mentions) Prewet filter mats, by PerkinElmer (2 mentions) Filter sample bags, by PerkinElmer (2 mentions) 96 well plate, by Corning (2 mentions) Anti cd3, by Thermo Fisher Scientific (1 mentions) Lymphoprep, by Abbott (1 mentions) Filtermate cell harvester, by PerkinElmer (1 mentions) Anti cd28, by BD (1 mentions) 7 aad, by BD (1 mentions) Sulforhodamine b srb dye, by Biotium (1 mentions) Glass fiber filters, by Brandel Inc (1 mentions) Scintiverse scintillation fluid, by Thermo Fisher Scientific (1 mentions) Tritiated thymidine, by MP Biomedicals (1 mentions) Scintillation counter, by PerkinElmer (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Concanavalin a (cona), by Merck Group (1 mentions)

Spelling variants of this product

Thymidine (11 citations) Tritiated thymidine (3H-TdR) (4 citations) Tritiated thymidine [3H]-thymidine (3 citations) Tritiated (3H-) thymidine (2 citations)

18 protocols using tritiated thymidine

Lymphocyte Proliferation Assay with Anti-CD3

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Lymphocytes were separated from whole blood using lymphoprep (Alere), and the cells were then cultured at a concentration of 10⁶/ml with 10% AB serum in RPMI with and without anti-CD3 at a final concentration of 0.3 µg/ml (eBioscience) in triplicate, then incubated at 37°C with 5% CO₂ for 4 d.
For both assays to measure the proliferation, 10 µl (1 μCur) of tritiated thymidine (PerkinElmer) was added to each of the wells, and the plate was returned to the incubator for 4 h. Any actively dividing cells incorporated the tritiated thymidine. The plates were then harvested (FilterMate cell harvester; PerkinElmer) and the radioactivity was counted using the β counter (TopCount β counter; PerkinElmer). The amount of proliferation was demonstrated by the amount of tritiated thymidine incorporation into the cells measured by CPM on the β counter. Control samples were always set up alongside patient samples. For PHA stimulation, control results should be <400 CPM for 0 µg/ml PHA and >12,500 for the highest CPM. For CD3 stimulation, control results should be <600 CPM without anti-CD3 and >6,000 with anti-CD3.

Standing A.S., Malinova D., Hong Y., Record J., Moulding D., Blundell M.P., Nowak K., Jones H., Omoyinmi E., Gilmour K.C., Medlar A., Stanescu H., Kleta R., Anderson G., Nanthapisal S., Gomes S.M., Klein N., Eleftheriou D., Thrasher A.J, & Brogan P.A. (2017). Autoinflammatory periodic fever, immunodeficiency, and thrombocytopenia (PFIT) caused by mutation in actin-regulatory gene WDR1. The Journal of Experimental Medicine, 214(1), 59-71.

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Evaluating Drug-Specific T Cell Responses

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PBMCs from patients were isolated with Ficoll-Paque, and 2 × 10⁵ cells were cultured for 5 days in complete medium and 5% heat-inactivated human serum AB in 96-well round-bottomed microwell plates with or without RTX (50 μg/ml) and TCZ (50–25-12.5 μg/well). For the assessment of drug specific T cell response, PBMCs of patient 2# were cultured in medium alone or TCZ with or without anti-major histocompatibility complex (MHC) class II (5 μg/ml) for 5 days.
Sixteen hours before harvesting, 0.5 µCi of Tritiated Thymidine (PerkinElmer, Boston, USA) was added to each well, and radionuclide uptake was measured by scintillation counting (MicroBeta TriLux PerkinElmer, Boston, USA). Mitogenic index (ratio between mean value of counts per minute—c.p.m.—of samples and medium alone, MI) ≥ 2 was considered positive as described^{17 (link)}.

Vultaggio A., Nencini F., Bormioli S., Silvestri E., Dies L., Vivarelli E., Maggi E, & Matucci A. (2021). Drug-specific Treg cells are induced during desensitization procedure for rituximab and tocilizumab in patients with anaphylaxis. Scientific Reports, 11, 12558.

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Investigating S-Ag Apitopes' Immunosuppressive Effects

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Example 4

The ability of the S-Ag apitopes to inhibit the immune response is investigated in healthy HLA-DRB1*DR3 mice ex vivo. Mice are pretreated with different apitopes according to the following schedules:

Mice are injected subcutaneously in the flanks with S-Ag peptides (100 μg/injection) or PBS at day −8, −6, −4 (high dose schedule). Alternatively, mice are injected using a dose escalation schedule, wherein e.g. X μg of peptide are administered, followed by 10×μg, then 100×μg then 1000×μg. For example on day 0, the mice may be injected subcutaneously in the base of the tail with 50-100 μg antigen/CFA (S-Ag or the native sequence of the tolerogenic peptide). Ten days after immunization, the draining LNs and spleens are harvested. Proliferation assay are then performed as described below.

Proliferation assay After 72 hours, 60 μL of cell supernatant are harvested and frozen. 20 μL/well of tritiated thymidine (PerkinElmer, Zaventem, Belgium) are then added to the cells to obtain a final concentration of 1 μCi/well. The cells are incubated at 37° C., and after 16 h, plates are frozen. Thawed plates are harvested and read with β-counter (Wallac 1450 Microbeta Trilux Liquid Scintillation Counter) to assess the cell proliferation.

US11542316B2. S-Arrestin peptides and therapeutic uses thereof (2023-01-03). APITOPE INTERNATIONAL NV [BE]. Inventors: David Wraith [GB], Evelien Schurgers [GB], Keith Martin [GB], Liselotte Jansson [GB].

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Suppressive Assay of Human Tregs

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Antigen-presenting cells, CD4⁺CD25⁻ T responder cells (Tresponders), and CD4⁺CD25⁺ Tregs were isolated from 2 healthy independent control patients, patient II.3, and his mother (patient I.2). Mixed lymphocyte reactions were performed using 5 × 10³ CD4⁺CD25⁻ Tresponders and 5 × 10⁴ irradiated (at 3300 rad) T-cell–depleted antigen-presenting cells isolated from healthy blood donors. Tregs were added to cell culture at titrations of 1:32 to 1:1. The culture medium was cRPMI supplemented with 10% human serum and 2.5 μg/mL of soluble anti-CD3 (UCHT1) and anti-CD28 (BD Biosciences). Proliferation was read (in a scintillation counter) at day 7 upon addition of 1 μCi tritiated thymidine (Perkin Elmer, Waltham, MA) for the last 18 hours of culture. The suppressive phenotype of CD25²⁺ cells was confirmed through in vitro suppression assays. Using Tregs from healthy donors, suppression of Tresponder proliferation at a 1:1 ratio is typically >75%.

Okou D.T., Mondal K., Faubion W.A., Kobrynski L.J., Denson L.A., Mulle J.G., Ramachandran D., Xiong Y., Svingen P., Patel V., Bose P., Waters J.P., Prahalad S., Cutler D.J., Zwick M.E, & Kugathasan S. (2014). Exome Sequencing Identifies a Novel FOXP3 Mutation in a 2-Generation Family With Inflammatory Bowel Disease. Journal of pediatric gastroenterology and nutrition, 58(5), 561-568.

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Evaluating Toxicity of Metal Oxide NPs

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A549 cells were obtained from the American Type Culture Collection (ATCC CCL-185, Manassas, VA, USA). The seven transition metal oxide NPs (TiO₂, Cr₂O₃, Mn₂O₃, Fe₂O₃, NiO, CuO, and ZnO) were purchased from Nanostructured and Amorphous Materials (Houston, TX, USA). Sulforhodamine B (SRB) dye was procured from Biotium (Freemont, CA, USA). Annexin V-FITC and 7-AAD were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Tritiated thymidine came from Perkin-Elmer (Waltham, MA, USA) and propidium iodide (PI) was purchased from Fisher Scientific (Pittsburgh, PA, USA).

Tolliver L.M., Holl N.J., Hou F.Y., Lee H.J., Cambre M.H, & Huang Y.W. (2020). Differential Cytotoxicity Induced by Transition Metal Oxide Nanoparticles is a Function of Cell Killing and Suppression of Cell Proliferation. International Journal of Molecular Sciences, 21(5), 1731.

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Splenocyte Mitogenesis Assay Protocol

Cited 1 time

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Splenocyte mitogenesis was performed following procedures described in
detail by Burchiel et al., 2009 (link). Spleen
cells were plated at 2 × 10⁵ cells (in replicates of 6/mouse)
in wells of a 96-well U-bottom plate. Splenocytes were stimulated with either 1
μg/mL concanavalin-A (Con-A; T-cell mitogen; Sigma Aldrich) or 10
μg/mL lipopolysaccharide (LPS; B-cell mitogen; Enzo Life Sciences) for 48
h. After 48 h simulation, cells were pulsed with 1 μCi tritiated
thymidine (Perkin Elmer) and incubated for 18 h. Cells from each well were then
harvested onto glass fiber filters (Brandel, Gaithersburg, MD) using a Brandel
Model M-96T cell harvester (Brandel, Gaithersburg, MD) and lysed with a 0.05%
(v/v) Tween-20 solution. Dried filter paper samples were then transferred into
liquid scintillation vials containing 3 mL ScintiVerse scintillation fluid
(Fisher Scientific) and allowed to sit at RT for 30 mins. Tritiated thymidine
incorporation was then assessed by liquid scintillation counting using a Beckman
Coulter LS 6500 Multi-Purpose Scintillation Counter (Beckman Coulter,
Indianapolis, IN).

Bolt A.M., Medina S., Lauer F.T., Liu K.J, & Burchiel S.W. (2019). MINIMAL URANIUM IMMUNOTOXICITY FOLLOWING A 60-DAY DRINKING WATER EXPOSURE TO URANYL ACETATE IN MALE AND FEMALE C57BL/6J MICE. Toxicology and applied pharmacology, 372, 33-39.

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Proliferation Assay for HUVECs

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4×10³ HUVECs were seeded and grown for 24 h, and quiescence was induced by incubating for 12 h in basal media + 0.1% BSA. HGF or NK1 variants were then added, along with 10 pM FGFb, and incubated for 24 h at 37 °C/5% CO₂. Next, 2 μCi tritiated thymidine (MP Biomedicals) was added to each well and incubated for an additional 24 h at 37 °C/5% CO₂, after which the supernatant was removed and the cells lysed via freeze-thaw. The amount of tritiated thymidine incorporated into newly synthesized DNA was measured using a scintillation counter (PerkinElmer). Error bars represent the standard deviation of triplicate wells. Data was measured against negative control with only FGFb added.

Liu C.J., Jones DS I.I., Tsai P.C., Venkataramana A, & Cochran J.R. (2014). An engineered dimeric fragment of hepatocyte growth factor is a potent c-MET agonist. FEBS letters, 588(24), 4831-4837.

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Canine Cell Proliferation Assay

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Canine whole blood was collected in heparin sulfate tubes from beagle dogs as well as dogs from mixed breeds. Whole blood (20 μL) was plated in 96-well plates (Corning Costar, Tewksbury, MA, USA) with 180 μL of DMEM complete medium (Dulbecco's modified Eagle medium; 10% heat-inactivated fetal bovine serum; 100 U/mL penicillin; 100 μg/mL streptomycin; Gibco Life Technologies, Grand Island, NY, USA) containing vehicle control or oclacitinib (0.001–10 μm), concanavalin A (ConA; 1 μg/mL; Sigma-Aldrich, St. Louis, MO, USA), and canine interleukin-2 (IL-2; 50 ng/mL; R&D Systems, Minneapolis, MN, USA). Plates were incubated at 37 °C for 48 h. Tritiated thymidine, 0.4 μCi per well (Perkin Elmer, Waltham, MA, USA), was added for 20 additional hours. Plates were frozen and then thawed, washed, and filtered using a Brandel MLR-96 cell harvester (Gaithersburg, MD, USA) and prewet filter mats (Perkin Elmer). Filters were dried and placed into filter sample bags (Perkin Elmer) with 10 mL of scintillant (Perkin Elmer). Sealed filters were counted on an LKB Wallac 1205 Betaplate liquid scintillation counter (Pharmacia, Uppsala, Sweden). Data were expressed as percent control, and dose–response data were then analyzed using a 4-parameter logistic equation.

Gonzales A.J., Bowman J.W., Fici G.J., Zhang M., Mann D.W, & Mitton-Fry M. (2014). Oclacitinib (APOQUEL®) is a novel Janus kinase inhibitor with activity against cytokines involved in allergy. Journal of Veterinary Pharmacology and Therapeutics, 37(4), 317-324.

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T-cell Proliferation Assay with CB-MSCs

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CB-MSCs were either untreated or treated with IFNγ (40 ng/ml). After 24 hours incubation, the cells were harvested, washed three times, and then used in the T-cell proliferation assay. The anti-CD3/CD28 proliferation assay was performed as previously described [20 (link)]. Isolated peripheral blood mononuclear cells (PBMCs) were reconstituted (at 100,000–200,000 cells/well in a 96-well-plate) with RPMI complete medium (RPMI 1640, supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine), stimulated to proliferate with anti-CD3/CD28 antibodies (6 μg/ml) (eBioscience, San Diego, CA) and cocultured with irradiated (3,000 rad) CB-MSC for 3 days. CD4 T lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) incorporation (CellTrace CFSE, Molecular Probes, Invitrogen, Carlsbad, CA) as previously described [20 (link)]. In other instances, lymphocyte proliferation was measured by radioactive thymidine incorporation in PBMC. For this, cells in the above-described assay were pulse-treated with tritiated thymidine (PerkinElmer, Waltham, MA) on the third day for 8–16 hours and thymidine incorporations were later analyzed with a liquid scintillation counter.

Mounayar M., Kefaloyianni E., Smith B., Solhjou Z., Maarouf O.H., Azzi J., Chabtini L., Fiorina P., Kraus M., Briddell R., Fodor W., Herrlich A, & Abdi R. (2015). PI3Kα and STAT1 Interplay Regulates Human Mesenchymal Stem Cell Immune Polarization. Stem cells (Dayton, Ohio), 33(6), 1892-1901.

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Nanoparticle Cytotoxicity Evaluation Protocol

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NiO NPs were purchased from Nanostructured and Amorphous Materials (Los Alamos, Houston, TX, USA) and Ni(OH)₂ NPs were purchased from US Research Nanomaterials (Houston, TX, USA). A549 cells and HepG2 cells were acquired from the American Tissue Culture Collection (Manassas, VA, USA). 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) and propidium iodide (PI) were obtained from Fisher Scientific (St. Peters, MO, USA). The JC-1 Mitochondrial Membrane Potential Detection Kit and sulforhodamine B were purchased from Biotium (Freemont, CA, USA). Ac-DEVD-pNA was obtained from Anaspec (Fremont, CA, USA). Annexin V-FITC and 7-aminoactinomycin D (7-AAD) were acquired from BD Biosciences (Franklin Lakes, NJ, USA). Tritiated thymidine was purchased from Perkin-Elmer (Waltham, MA, USA). Other chemicals used for experiments were of the highest purity that could be obtained.

Cambre M.H., Holl N.J., Wang B., Harper L., Lee H.J., Chusuei C.C., Hou F.Y., Williams E.T., Argo J.D., Pandey R.R, & Huang Y.W. (2020). Cytotoxicity of NiO and Ni(OH)2 Nanoparticles Is Mediated by Oxidative Stress-Induced Cell Death and Suppression of Cell Proliferation. International Journal of Molecular Sciences, 21(7), 2355.

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