RBC Antibody – Rabbit

Rabbit

1.0 ml in PBS+ 0.02% NaN3

Stable at 4°C. For long term storage, aliquot and freeze unused portions at -20°C in volumes appropritate for single usage. Avoid repeated freeze/thaw cycles.

This reagent is not sold as sterile but may be sterilized by filtration if necessary. To minimize loss of volume during filtration, dilute to the final working concentration in the appropriate medium before filtration and filter through a 0.22um Millipore filter (or equivalent).

1. Wash and pack sheep red blood cells in VBD media. Dilute to 1 % in VBD.
2. Using a 96-well round-bottom microtitre plate, add the antibody to a final
concentration of 1:2 and dilute serially to a dilution of 1:2 million using doubling dilutions.
3. Add cells and incubate for 30 minutes at room temperature.
4. Centrifuge to pellet the cells and discard the supernatant.
5. Resuspend to the original volume in MBSs Hemo-Lo Guinea Pig Complement diluted to 1:20 in VBD.
6. Incubate for 60 minutes at 37°C.
7. Read plate by visual inspection to determine hemolytic titre.

Rabbit anti-sheep red blood cell stroma

For haemolytic assays using 1-2% srbc's

The suggested dilution is 1:400.
For complement fixation the appropiate dilution should be determined in house.