Hemolysis test: refers to the hemolysis and red blood cell aggregation caused by pharmaceutical preparations. Hemolytic reactions include immune hemolysis and non-immune hemolysis.
1. Prepare 2% Red Blood Cells
Baoding rabbits, draw blood from the heart 5 m, add 0.2 ml of anticoagulant EDTA, wash with PBS, centrifuge to remove the white blood cells on the surface, until the supernatant does not show red, discard the supernatant, take 1 ml and add 50 ml of PBS.
2. Prepare Materials
Adjust the concentration of different kinds of materials to 800, 400, 200, 100, 50 and 25 ug/mb with PBS solution, and the concentration of gold clusters to 50, 25, 12.5, 6.25, 3.15 ug/ml.
3. Experimental Process
Before each use of red blood cells, wash with PBS until there is no obvious red (slightly yellow) in the supernatant, and draw 200ul of the substrate. Release to 10ml for use.
Mix 0.5ml of the material with 0.5ml of 2% red blood cell solution, stand still at room temperature for 3 hours, and then centrifuge at 10050 r/min for 3 minutes. Take out 100ul to 96-well plate, use 570nm wavelength to detect. Water and PBS were used as positive and negative controls, respectively. Two parallels for each sample.
Hemolysis rate (%) = (sample absorption-negative control absorption) / (positive control absorption-negative control absorption) x 100%. A hemolysis rate of more than 5% is regarded as hemolysis.
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