Interferon gamma release assay (IGRA) was performed as previously described [7 (link),12 (link),16 (link)]. Briefly, 0.75 ml of heparinized blood diluted 1:1 in RPMI medium (Fisher, Hampton, USA) supplemented with 1% sodium heparin (Roche, Basilea, Switzerland) and 1% penicillin/streptomycin (pen/strep) (Fisher, Hampton, USA) was stimulated with antigens: bovine or avian tuberculins (PPD-B and PPD-A, respectively, CZ Vaccines, Porriño, Spain) at a final concentration of 30 μg/ml; pokeweed mitogen (PWM) at a final concentration of 5 μg/ml (Sigma-Aldrich, St. Louis, USA); CFP-10/ESAT-6 (CE) (Lionex, Braunschweig, Germany) cocktail at a final concentration of 5 μg/ml protein; M. bovis P22 complex at a concentration of 10 μg/ml or without antigen (nil negative control) (RPMI + Pen/strep), and kept at 37 °C with 5% CO2 for 16–20 h. Post-incubation, supernatants were collected in duplicate aliquots (250 μl) and stored at −80 °C. IFN-γ levels were assessed by sandwich ELISA using anti-badger IFN-γ capture monoclonal antibody 10H6–C1 and biotinylated monoclonal antibody 11B9 (Animal and Plant Health Agency, UK). Optical density (OD) was measured with 450 nm filters by ELISA reader. The cut-off point of 0.044 (using combined PPD-B and PPD-A values) was used to identify positivity to M. bovis infection [16 (link)].
Sodium heparin
Manufactured by Roche
Sourced in Brazil, Switzerland
Sodium heparin is an anticoagulant used in laboratory settings to prevent blood clotting in collected samples. It inhibits the blood coagulation cascade, allowing for accurate analysis of blood components.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Merck Group (3 mentions)
Penicillin streptomycin pen strep, by Thermo Fisher Scientific (2 mentions)
Rpmi medium, by Thermo Fisher Scientific (2 mentions)
Xylene, by Merck Group (2 mentions)
Ethanol, by Merck Group (2 mentions)
Pokeweed mitogen pwm, by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Vitamin e, by Merck Group (1 mentions)
Pokeweed mitogen, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Rosiglitazone, by Cayman Chemical (1 mentions)
Gw9662, by Cayman Chemical (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Ouabain, by Merck Group (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
6 protocols using sodium heparin
1
Interferon Gamma Release Assay for Mycobacterium bovis
2
Antioxidant and Anti-inflammatory Protocol
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Sodium citrate, AEC, NAC, vitamin E, dexamethasone, and hydrogen peroxide were purchased from Sigma Chemical Co. (Saint Louis, MO, USA); ethanol, methanol, and xylene from Merck (Rio de Janeiro, RJ, Brazil); and sodium heparin from Roche (São Paulo, SP, Brazil). All solutions were freshly prepared immediately before use.
3
Badger IFN-γ ELISA for M. bovis Infection
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IGRA was performed as previously described (21 (link)); 1.5 ml aliquots of heparinized blood diluted 1:1 in RPMI medium (Fisher, Hampton, USA) supplemented with 1% sodium heparin (Roche, Basilea, Switzerland) and 1% penicillin/streptomycin (pen/strep) (Fisher, Hampton, USA) was stimulated with a final concentration of 30 μg/ml bovine and avium tuberculins (Spanish PPD-B and PPD-A, respectively, CZ Vaccines, Porriño, Spain), with pokeweed mitogen at a final concentration of 5 μg/ml (Sigma-Aldrich, St. Louis, USA), with CFP-10/ESAT-6 (Lionex, Braunschweig, Germany) at a final concentration of 5 μg/ml protein cocktail or without antigen (RPMI+Pen/strep), and kept at 37°C with 5% CO2 for a minimum of 16 h. Post-incubation, supernatants were collected in duplicate aliquots (250 μl) and stored at −80°C. IFN-γ levels were assessed by sandwich ELISA using anti-badger IFN-γ capture monoclonal antibody 10H6-C1 and biotinylated monoclonal antibody 11B9 (Animal and Plant Health Agency, UK). The cut-off point of 0.044 (using combined PPD-B and PPD-A values) was used to identify positivity to M. bovis infection.
4
Reagent Acquisition for Protocol
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Dimethyl sulfoxide (DMSO) was purchased from Sigma Chemical Co. (Saint Louis, MO, USA), rosiglitazone and GW9662 from Cayman Chemicals (Saint Louis, MO, USA), ethanol, methanol and xylene from Merck (Rio de Janeiro, Brazil) and sodium heparin from Roche (São Paulo, Brazil). All solutions were freshly prepared immediately before use.
5
Isolation and Culture of Human PBMCs
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The ethics committee of the Hospital Universitário Clementino Fraga Filho-UFRJ agreed with the study protocol, which is registered under the approval number 148/09. Blood samples were obtained from peripheral blood samples collected from healthy volunteers using sodium heparin (Roche, Rio de Janeiro, Brazil) as anticoagulant. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation with Ficoll-Paque (GE, USA). PBMC were seeded at a concentration of 5 × 106 cells per well (1 mL final volume) for 2 hours in 24-well plates (TPP, Switzerland), in humidified chamber with 5% CO2 atmosphere at 37°C. Afterwards, nonadherent cells were removed by extensive washing. Adherent cells were cultured in RPMI1640 medium (Sigma, Chemical Co., USA) supplemented with 10% fetal bovine serum (FBS, 500 μL final volume) with or without 50 ng/mL GM-CSF (PeproTech, USA) and 50 ng/mL IL-4 (R&D Biosystems, USA), for 5 days. In some experiments, cells were preincubated with 100 nM Ouabain (Sigma, Chemical Co., USA) for 24 h, followed by the addition of fresh medium or the cytokines described above.
6
Cardiovascular Pharmacological Interventions
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Captopril, Olmesartan, CGP42112A, and alloxan monohydrate were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). PD123319 from Cayman Chemical Co. (Ann Arbor, MI, USA). Ethanol, methanol, and xylene from Merck (Rio de Janeiro, RJ, Brazil). Sodium heparin and sterile saline solution from Roche (São Paulo, SP, Brazil). All solutions were prepared immediately before use.
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