Hello,
Could you please guide me with a standard protocol to do a hemolysis test for biomacromolecules?
thanks in advance!
Ajeet Kumar
- Human and rat (Sprague–Dawley) blood samples were purchased by Bioreclamation IVT (Westbury, NY, USA), collected in tubes containing heparin as anticoagulant and stored at 4 °C before use. Dog and bovine whole blood was collected from in-house animals at Zoetis Animal Health, Kalamazoo, Michigan and stored at 4 °C in tubes containing heparin as anticoagulant. 10 mL of each whole blood sample were centrifuged at 1,500 × g for 5 min and the resulting plasma fraction was removed from the samples. The pellets were washed with an equal volume (10 mL) of saline, mixing by inversion. The centrifuging and washing steps were repeated 5 times. After the last washing step, a volume of PBS was added, and the resulting samples were diluted 1:10 with PBS to yield an RBC concentration of ~ 5 × 108 RBC/mL. The assay was performed in a 384-well polypropylene microplate (Greiner 784201, Greiner Bio-One International, GMBH) through a Bravo Liquid Handling Platform 384 system (Agilent Technologies, Santa Clara, CA, USA) according to the previously published procedures29. The compounds were tested in serial 1:1 dilutions in PBS, with final concentrations ranging from 150 to 1.2 µM. Melittin was used as reference lytic peptide. 1% TritonX100 and PBS were used as positive and negative (diluent) controls, respectively. The final concentrations were obtained by adding to the wells 7 µL of each solution, melittin or PBS, followed by 63 µL of RBCs to each well. The plate was sealed, shaken on a plate shaker for 20 s, incubated for one hour at 37 °C. Finally, it was centrifuged at 1,500 × g for 5 min at room temperature. 60 µL of supernatants were removed from all plate wells via a Bravo 384 robot and transferred to a flat-bottomed 384-well plate (Greiner UV-Star #7810801, Greiner Bio-One International, GMBH). The plate was centrifuged at 1,000 g for 1 min to remove air bubbles and optical density was measured at 405 nm using a plate reader.
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