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.