pFRT

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	The pFRT expression vector containing the cMBL encoding sequence was transfected into 80% confluent Hela R19 cells using polyethyleneimine (PEI) (Polysciences, USA) in a 1:5 ratio (μg DNA: μg PEI). At 12 h post transfection, the transfection mixture was replaced by 293 SFM II expression medium (Gibco), supplemented with GlutaMax (10 ml/liter, Gibco), primatone (3 g/l, Sigma − Aldrich, USA), d-glucose (2 g/l, Sigma − Aldrich), sodium bicarbonate (3.7 g/l, Merck, Germany), DMSO (15 ml/l, Sigma − Aldrich), penicillin (100 units/ml) and streptomycin (100 μg/ml).

Protocol tips

The pFRT expression vector containing the cMBL encoding sequence was transfected into 80% confluent Hela R19 cells using polyethyleneimine (PEI) (Polysciences, USA) in a 1:5 ratio (μg DNA: μg PEI). At 12 h post transfection, the transfection mixture was replaced by 293 SFM II expression medium (Gibco), supplemented with GlutaMax (10 ml/liter, Gibco), primatone (3 g/l, Sigma − Aldrich, USA), d-glucose (2 g/l, Sigma − Aldrich), sodium bicarbonate (3.7 g/l, Merck, Germany), DMSO (15 ml/l, Sigma − Aldrich), penicillin (100 units/ml) and streptomycin (100 μg/ml).

Publication protocol

HeLa R19 cells, a gift from Dr. Wentao Li (Utrecht University, The Netherlands), were used to produce RcMBL. The cells were maintained in a humidified 37 °C incubator with 5% CO2, and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, United Kingdom) supplemented with 10% Fetal Bovine Serum (FBS) (Bodinco BV, The Netherlands), penicillin (100 units/ml) and streptomycin (100 μg/ml). The pFRT expression vector containing the cMBL encoding sequence was transfected into 80% confluent Hela R19 cells using polyethyleneimine (PEI) (Polysciences, USA) in a 1:5 ratio (μg DNA: μg PEI). At 12 h post transfection, the transfection mixture was replaced by 293 SFM II expression medium (Gibco), supplemented with GlutaMax (10 ml/liter, Gibco), primatone (3 g/l, Sigma − Aldrich, USA), d-glucose (2 g/l, Sigma − Aldrich), sodium bicarbonate (3.7 g/l, Merck, Germany), DMSO (15 ml/l, Sigma − Aldrich), penicillin (100 units/ml) and streptomycin (100 μg/ml). The expression medium was harvested at 5 days post transfection (de Vries et al., 2010, Longo et al., 2013).

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