Haemolysis assay

Sangue intero raccolto (topo anestetizzato con etere) in una provetta contenente Alsever's solution (indicativamente diluito 1:10)

• • Centrifugare 10 min 1500 rpm Falcon 15 ml (pellet=globuli rossi: togliere anello polimorfonucleati)

  • 2 lavaggi in PBS o Plaque Buffer(P.B.)nel caso di Np-Kephalyn (dopo il primo lavaggio contare i globuli rossi)

  • Aggiungere un ugual volume (del pellet) di NP-BSA diluito in PBS (5mg/ml 1:1) oppure 600 mg/ml NP-Kephalyn diluito in P.B.(gift of M.S. Neuberger, Cambridge, UK)

  • 30 min 37°C, 2h nel caso di NP-Kephalyn

  • 3 lavaggi in PBS o P.B. e risospendere in PBS Mg⁺⁺ Ca^{++ (globuli rossi al 2% finale)}, 50µl globuli rossi 2%/campione in Eppendorf 500µl

  • Aggiungere 10ng, 100 ng di IgM (AS) tutti i punti con uguale volume in PBS Mg⁺⁺ Ca⁺⁺

  • Incubare 10 min 37°C

  • Aggiungere 5µl di Guinea Pig Complement 1:2

  • Incubare 10 min 37°C

  • Centrifugare 5 min 2000 rpm centrifuga Eppendorf

  • Aggiungere 100µl di Guinea Pig Complement 1:2

  • Mettere 100µl di campione in ogni pozzetto di una piastra da ELISA ed effettuare una lettura a 405 nm

    • SOLUZIONI

  • PBS1X

  • PBS 1X Mg⁺⁺Ca⁺⁺: PBS 10ml + 100µl MgCl₂ 0,1M + 100µl CaCl₂ 0,1M

  • Alsever's solution: 0,42% NaCl, 0,8% trisodio citrato, 2,05 % glucose pH6.1 con acido citrico 10%)

  • Plaque Buffer: Medium 199 10ml +100µl MgCl₂ 0,1M + 120µl NaPO₄ 0,4M

Haemolysis assays

Red blood cells (RBC) were obtained from BALB/c mice according to the Institutional Animal Care guidelines, pelletted and stored in Alsever’s solution at 4°C. Before use, the RBC were washed three times and then incubated for 2 h at 37°C with 600 mg/ml of NP-cap-kephalyn (a kind gift of M.S. Neuberger, Cambridge, UK) in TC199 medium supplemented with 1 mM MgCl2, 1mM CaCl2. RBC were then washed three times in PBS and diluted 1:50 in TC199 medium supplemented with 1 mM MgCl2, 1mM CaCl2. After 10 min of incubation at 37°C of 25 ml of NP-coated RBC suspension with 25ml of IgM at different concentrations (from 20 to 2000 ng), 5 ml of guinea pig complement (Sigma Chemical Company, St.Louis, Missouri, U.S.A.) were added. After 10min at 37°C, hemolysis was quantitated by spectrophotometric measurements (absorbance at 405 nm) of the supernatants diluted in 100 ml of PBS.