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, See Table 2 for volume recommendation

, Resuspend the beads in the vial (i.e. vortex for >30 sec, or tilt and rotate for 5 min)

, Add the same volume of Buffer A or B, or at least 1 mL, and resuspend

, Remove the tube from the magnet and resuspend the washed beads in the same volume of Buffer A or B as the initial, vol.of beads

, Couple Ligands to the Dynabeads ®

, ? This protocol is based on 5 mg (~165 ?L) Dynabeads ® M -280 Tosylactivated. It is not recommended to couple < 5 mg beads at a time. For larger volumes than 10 mg beads

, The ligand volume (?L) is dependent on the ligand concentration (?g/uL) and must be calculated for each ligand, e.g. when coupling 5 mg beads, the Ab requirement is 100 ?g, ? Use 100 ?g ligand/5 mg beads

, It is decreased to 20 mg/mL when coupling the smallest amount (5 mg beads), to allow for a sufficient volume for efficient mixing

, Transfer 165 ?L washed and resuspended beads to a new tube, place in a magnet for 1 min, and remove the supernatant

, Resuspend the beads in 100 ?g ligand* and add Buffer A (or B) to give a total volume of 150 ?L**. Mix thoroughly by vortex or pipetting

, Remove the tube from the magnet and add 1 mL Buffer E, vortex for 5-10 sec

, NS1 cells were cultivated in Dulbecco's medium with 15% of foetal bovine serum, 1% of non-essential amino acids, 1% of antibiotics (penicillin and streptomycin) and 1% of L-glutamine at 37 ? C under a controlled atmosphere containing 7% of CO 2 . They were centrifuged at 1000 RPM (centrifuge diameter 344 mm) for 10 min at 9 ? C and then diluted in PBS (Dulbecco's Phosphate Buffer Saline, Gibco, Life Technologies) in which the tests were carried out. CHO cells were cultivated in Ham F-12 Nutrient Mixture with 10% of foetal bovine serum

, ? C under a control atmosphere containing 5% of CO 2 . They were washed two times in PBS, let in a solution of 0.25% trypsin-EDTA for 5 min at 37 ? C and were centrifuged at 1000 RPM (centrifuge diameter 344 mm) for 5 min at 9 ? C. Finally

, Up to eight mice were kept in each cage and housed in a temperature-regulated-room and had free access to food and water. All animals' experiments were performed to minimize suffering according to the guideline of the CETEA committee, 2001.

, Ipad gene was amplified from Shigella flexneri (CIP 82.48T) and cloned into the IPTG inducible pET22b(+) vector (Novagen) allowing insertion of a poly-histidine tag sequence at the 3' end of the gene used for protein purification. Hybridomas were produced by fusing spleen cells of immunized mice with NS1 myeloma cells

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