Spleens were harvested from each mouse on the indicated day post-sensitization. Mononuclear cells were isolated with Histopaque-1083 (Sigma). Cells were resuspended at 1 × 106 cells/ml in complete media [RPMI-1640 media (Mediatech, Manassas, VA, USA) supplemented with 1% L-glutamine (Mediatech), 1% antibiotics (Mediatech), 50 μM 2-mercaptoethanol (Sigma) and 10% Cosmic calf serum (Hyclone, Logan, UT, USA)]. Next, 100 μl of cells were added to each well of a 96-well round-bottomed plate (Corning, Corning, NY, USA). PLP139–151 in 100 μl of complete media was added into culture in a dose dependent manner. Cells were incubated at 37°C, 5% CO2 for the indicated times in the presence of the indicated peptide doses. Anti-IGF-1R antibody (αIR3) (Millipore, Marlborough, MA, USA) was added into the PLP139–151-stimulated spleen cell cultures at a final concentration of 1 μg/ml. Eighteen hrs prior to harvesting cultures, the cells were pulsed with 1 μCi/well of tritiated thymidine (3H-TdR) (PerkinElmer, Boston, MA, USA). The cells were harvested onto glass fiber filters (PerkinElmer) for measurement of radiolabel incorporation using a liquid scintillation counter (PerkinElmer).
Tritiated thymidine 3h tdr
Manufactured by PerkinElmer
Sourced in United States
Tritiated thymidine (3H-TdR) is a radioactive nucleoside analog that is commonly used as a tracer in cell proliferation studies. It is an isotopically labeled form of the DNA base thymidine, with the hydrogen atom at the 5 position of the thymine ring replaced by the radioactive tritium (3H) isotope. This radioactive label allows researchers to track the incorporation of thymidine into newly synthesized DNA, providing a measure of cell division and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Glass fiber filter, by PerkinElmer (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Liquid scintillation counter, by PerkinElmer (2 mentions)
3h tdr, by PerkinElmer (2 mentions)
Histopaque 1083, by Merck Group (1 mentions)
Round bottomed 96 well plate, by Corning (1 mentions)
Tricarb liquid scintillation analyser, by PerkinElmer (1 mentions)
Mouse igg2a κ isotype control, by BD (1 mentions)
Mouse anti human hla dr, by BD (1 mentions)
Pancoll, by PAN Biotech (1 mentions)
4 protocols using tritiated thymidine 3h tdr
1
Spleen Cell Proliferation Assay
2
Murine Spleen Leukocyte Proliferation Assay
3
Allogeneic Lymphocyte Proliferation by DCs
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To detect the function of DCs in stimulating allogeneic lymphocyte proliferation in vitro, human monocyte-derived DCs were co-cultured with PBMCs obtained from another healthy donor at a 1:65 ratio in 96-well plates in RPMI 1640 medium containing rhGM-CSF, rhIL-4, and 10% FBS for an additional 5 days. Tritiated thymidine (3H-TdR, PerkinElmer, Waltham, MA, USA) was added at 1 μCi/well on day 4, and the cells were collected and measured using a TriCarb Liquid Scintillation Analyser (PerkinElmer) 12–18 hours later.
In the MHC Class II antibody competitive blocking experiment, 5 μL/mL of purified mouse anti-human HLA-DR (BD Biosciences) or mouse IgG2a, κ isotype control (BD Biosciences) was added to the DC-PBMC co-culture system, and 1 μCi/well 3H-TdR was added on day 4 and measured using a TriCarb Liquid Scintillation Analyser (PerkinElmer) 12–18 hours later.
In the MHC Class II antibody competitive blocking experiment, 5 μL/mL of purified mouse anti-human HLA-DR (BD Biosciences) or mouse IgG2a, κ isotype control (BD Biosciences) was added to the DC-PBMC co-culture system, and 1 μCi/well 3H-TdR was added on day 4 and measured using a TriCarb Liquid Scintillation Analyser (PerkinElmer) 12–18 hours later.
4
PBMC Proliferation Assay with Recombinant Proteins
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Human peripheral‐blood mononuclear cells (PBMC) were isolated from fresh blood of ten healthy volunteers using Pancoll (PAN‐Biotech, Aidenbach, Germany) density gradient centrifugation. 105 cells per well were cultured in 200 μL R10F medium (RPMI1640 supplemented with 200 U/mL penicillin, 200 μg/mL streptomycin, 4 mM L‐glutamine and 10% v/v fetal calf serum). Recombinant proteins were serially diluted to final concentrations of 25 μg/mL to 0.006 μg/mL and used to stimulate PBMCs. The recall antigen tetanus toxoid (Statens Serum Institute, Copenhagen, Denmark) was used as an additional control. Incubation in flat‐bottom 96‐well plates was performed for 7 days at 37°C in a humidified atmosphere of 5% CO2. Afterwards, 0.5 μCi/well of tritiated thymidine (3H‐TdR) (PerkinElmer, Boston, USA) was added to the cell culture and incubation was continued for a further 17 h. Proliferation of PBMCs was measured by 3H‐TdR incorporation 25 . All measurements were performed in triplicates, and the means are shown.
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