Gvbe buffer

Sensitized rabbit erythrocytes (Complement Technology Inc., Tyler, TX, USA) were washed once with tris buffered saline (TBS), centrifuged (3 min, 4°C, 500 g) and erythrocytes were re-suspended in GVB⁰ buffer (without Ca²⁺ and Mg²⁺, pH 7.3) (Complement Technology Inc., Tyler, TX, USA). GVB⁰ buffer contains 0.1% gelatin, 5 mM Veronal, 145 mM NaCl, and 0.025% NaN₃. GVB⁰ is a basic buffer which can be used to make other traditional buffers for complement assays. In order to determine 50 and 100% lysis, the erythrocytes were diluted with ddH₂O. The optical density was measured at 415 nm and OD values for 50 and 100% lysis were determined.
Serial dilution of porcine serum from 1:2 through 1:32 was prepared in GVB⁰ buffer with 5 mM MgEGTA. The addition of MgEGTA allows the process of complement activation via the alternative pathway. Then, sensitized rabbit erythrocytes were added to the diluted serum samples and incubated for 30 min at 37°C. Afterwards, ice cold GVBE buffer (with EDTA, pH 7.3) (Complement Technology Inc., Tyler, TX, USA) was added to the samples and samples were centrifuged (3 min, 4°C, 500 g). GVBE buffer contains 0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, and 10 mM EDTA, which inhibits the complement activation cascade. Afterwards, supernatant was transferred and optical density from supernatant was measured at 415 nm.

Sensitized sheep erythrocytes (Complement Technology Inc., Tyler, TX, USA) were washed once with tris buffered saline (TBS), centrifuged (3 min, 4°C, 500 g) and erythrocytes were re-suspended in GVB⁺⁺ buffer (with Ca²⁺ and Mg²⁺, pH 7.3) (Complement Technology Inc., Tyler, TX, USA). GVB⁺⁺ buffer contains 0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, 0.15 mM CaCl₂, and 0.5 mM MgCl₂, which allows the process of complement activation via the classical pathway. In order to determine 50 and 100% lysis, the erythrocytes were diluted with ddH₂O. The optical density was measured at 415 nm and OD values for 50 and 100% lysis were determined.
Serial dilution of porcine serum from 1:20 through 1:640 was prepared in GVB⁺⁺ buffer (with Ca²⁺ and Mg²⁺, pH 7.3). Then, Sensitized sheep erythrocytes were added to the diluted serum samples and incubated for 30 min at 37°C. Afterwards, ice cold GVBE buffer (with EDTA, pH 7.3) (Complement Technology Inc., Tyler, TX, USA) was added to the samples and samples were centrifuged (3 min, 4°C, 500 g). GVBE buffer contains 0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN₃, and 10 mM EDTA, which inhibits the complement activation cascade. Afterwards, the supernatant was transferred and optical density from supernatant was measured at 415 nm.