Hemolysis Assay
Wash and concentrate blood , prepare 2 % blood solution .
Add 3 mL rabbit blood to 14 mL PBS pH 5.7 and mix .
Centrifuge 10 min 500g 4C .
Remove supernatant .
Repeat steps for a total of 4 washes .
After the final wash , remove 200 uL blood from the bottom of the vial and add to 9.8 mL PBS pH 5.7 .
Prepare endosomal disruptor solutions .
Prepare 100 uL solutions of endosomal disruptors at different concentrations ( Ex serial 10x dilutions of TritonX from 15 mg / mL to . 0015 mg / mL ) .
Add blood to endosomal disruptors and incubate .
Add 50 uL blood solution to 100 uL endosomal disruptor solutions .
Incubate at 37C for 30 min , starting timer as soon as last solution added .
Make positive control .
Make a positive control ( known lysis ) by adding 50 uL blood solution to 100 uL diH2O and freeze - thaw cycling 3 times ( freeze in - 80C freezer ) .
Centrifuge and place in plate .
Centrifuge in tabletop centrifuge at 2500g for 6 min .
Carefully collect 75 uL of supernatant from each tube and place in 96 well plate .
* * Solutions bubble easily so be very carefully not to introduce any air * * .
Disrupting bottom pellet creates false positives .
Take absorbance .
Take absorbance at 541 nm .
Can also do absorbance scan if worried about side reactions
