PBMCs were isolated from heparinized blood samples of patients with grass pollen allergy by means of Ficoll density gradient centrifugation (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). PBMCs (2 × 105) were cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μL of serum-free Ultra Culture medium (BioWhittaker, Rockland, Me) supplemented with l -glutamine (10 mL of 200 mmol/L l -glutamine per liter; Sigma, St Louis, Mo), β-mercaptoethanol (5 mL of 50 mmol/L β-mercaptoethanol per liter, Sigma) and gentamicin (2 mL of 50 mg/mL per liter of medium, Sigma) at 37°C and 5% CO2 in a humidified atmosphere. PBMCs were stimulated with a mix of rPhl p 1, 2, 5, and 6 (0.5 μg per well of each allergen, recombinant mix) or rBet v 1 (2 μg per well) as a control in the presence of heat-inactivated (56°C for 30 minutes) 1:10 diluted sera obtained from rabbits 1 month after the last immunization with BM32 (40-μg dose), Alutard, or Allergovit or from normal rabbits. For control purposes, 4 U of IL-2 per well (Boehringer, Mannheim, Germany) or medium alone was used.
Nunclone
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
Nunclone is a laboratory equipment product designed for cell culture applications. It is a tissue culture surface that supports the growth and proliferation of various cell types. The core function of Nunclone is to provide a suitable substrate for cell attachment and propagation in in-vitro cell culture experiments and studies.
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Lab products found in correlation
2 protocols using nunclone
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Allergen-Specific PBMC Stimulation Assay
2
Allergen-Specific PBMC Stimulation Assay
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PBMCs were isolated from heparinized blood samples of patients with grass pollen allergy by means of Ficoll density gradient centrifugation (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). PBMCs (2 × 105) were cultured in triplicates in 96-well plates (Nunclone; Nalge Nunc International, Roskilde, Denmark) in 200 μL of serum-free Ultra Culture medium (BioWhittaker, Rockland, Me) supplemented with l -glutamine (10 mL of 200 mmol/L l -glutamine per liter; Sigma, St Louis, Mo), β-mercaptoethanol (5 mL of 50 mmol/L β-mercaptoethanol per liter, Sigma) and gentamicin (2 mL of 50 mg/mL per liter of medium, Sigma) at 37°C and 5% CO2 in a humidified atmosphere. PBMCs were stimulated with a mix of rPhl p 1, 2, 5, and 6 (0.5 μg per well of each allergen, recombinant mix) or rBet v 1 (2 μg per well) as a control in the presence of heat-inactivated (56°C for 30 minutes) 1:10 diluted sera obtained from rabbits 1 month after the last immunization with BM32 (40-μg dose), Alutard, or Allergovit or from normal rabbits. For control purposes, 4 U of IL-2 per well (Boehringer, Mannheim, Germany) or medium alone was used.
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