Four weeks after the last immunization, mice were euthanized and their spleens removed under aseptic conditions. The splenocytes were cultured, at a concentration of 4 × 106 viable cells/ml (100 µl per well), at 37°C under 5% CO2 in a 96-well flat-bottom plate (Nunc, Denmark), previously sensitized with 2 µg/ml or 10 µg/ml of recombinant proteins (rMEB), or 2 µg/ml or 10 µg/ml CBP. Splenocytes were cultured in RPMI 1640 medium (Thermo Fisher Scientific, MA, USA) supplemented with 10% heat-inactivated fetal calf serum (GIBCO BRL), penicillin–streptomycin (50 UI of penicillin; 50 µg/ml streptomycin), and amphotericin B (0.25 µg/ml). After 72 h, cells were pulsed for 8 h with 0.4 μCi thymidine (50 μCi/mmol; Amersham, UK) per well and the radioactivity incorporated in the DNA measured using a scintillation counter. Concanavalin A (ConA) (Sigma Aldrich, MO, USA), at a concentration of 10 µg/ml was used as proliferative positive control and 10 µg/ml albumin protein or 10 µg/ml total E. coli protein (CEP) were used as proliferative negative control. Cell proliferation data were expressed as the stimulation index of triplicate cultures from a cell pool from each group. These were obtained by dividing the amount of 3H-thymidine incorporated (c.p.m.) in antigen-stimulated cell cultured by the c.p.m. obtained from cells cultured without antigen (38 (link)).
Thymidine
Manufactured by Cytiva
Sourced in United Kingdom
Thymidine is a nucleoside that is a component of DNA. It is used in various laboratory applications, including cell culture, molecular biology, and biochemical research.
Automatically generated - may contain errors
Lab products found in correlation
3h thymidine, by Cytiva (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
96 well flat bottom plate, by Thermo Fisher Scientific (1 mentions)
3 protocols using thymidine
1
In Vitro Splenocyte Proliferation Assay
2
Generating Antigen-Specific T Cell Lines
3
Bovine Granulosa Cell Proliferation Assay
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Primary bovine granulosa cells were cultured for 24 h in McCoy's 5A medium and 10% fetal bovine serum (FBS). Cells were plated in 24-well plates (2 × 10 5 viable cells/well) and four replicates were tested for each experimental condition (APLN and/or IGF1 in the absence or in the presence of ML221) for each culture. After several washes and overnight serum starvation, cells were cultured for 24 h with 1 µCi/ µL [ 3 H] Thymidine (Amersham Life Science) in the presence or absence of APLN 13 or APLN 17 and/or IGF1 (10 -8 M).
Thymidine was then removed with PBS and cells were fixed with cold 50% trichloroacetic acid for 15 min on ice. Finally, cells were lysed using 0.5 N NaOH and the radioactivity was counted in a β-photomultiplier by adding scintillation fluid (Packard Bioscience).
Thymidine was then removed with PBS and cells were fixed with cold 50% trichloroacetic acid for 15 min on ice. Finally, cells were lysed using 0.5 N NaOH and the radioactivity was counted in a β-photomultiplier by adding scintillation fluid (Packard Bioscience).
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