Measurement of the red blood cell lysis by bacterial hemolysin
Lab/Group: Jeak Ling DING’s lab (National University of Singapore)
Related Journal & Article Information
Journal: Nature Immunology
Introduction
The lysis of the red blood cells can be monitored by the release of hemoglobin to the extracellular environment. After removing the intact RBC by centrifugation, the absorbance at 404 nm of the supernatant reflects the hemolysis. The RBC lysate obtained by total disruption by 1% Triton- X100, is used as a positive control.
Materials
Reagents
Equipment
Procedure
The RBC lysis by bacterial hemolysin is measured as described1 with modification.
1. Collect the RBCs into heparinized tubes.
2. Wash the RBC two times by gentle resuspension with 10 times volume of pyrogen free saline (0.9% NaCl), and centrifugation at 1000 x g for 10 min at room temperature.
3. Gently resuspend the RBC pellet with pyrogen free saline and dilute the RBC to 0.8% (v/v).
4. Culture the bacteria under test according to protocol #4, and adjust the population to 107 cfu/ml.
5. Incubate the washed RBC with equal volume of bacteria at room temperature under steady rotation at 6 rpm.
6. Set up pyrogen-free saline (0.9%) as the negative control, and 1% Triton-X100 in saline as the positive control.
7. At time intervals, retrieve aliquots of the reaction mixture and pellet the RBC by centrifuging at 1000 x g for 10 min at room temperature.
8. Transfer the supernatant into 96 well microtitre plate. Monitor the absorbance at 404 nm with ELISA-reader as an indication of the hemoglobin released into the supernatant.
Troubleshooting
Critical Steps
Anticipated Results
Hemolysin producing microbes, such as S. aureus, lyses the RBC and the typical A404= 1.2 – 1.6.
References
1. Shin, S. Y., Kang, J. H. & Hahm, K. S. Structure-antibacterial, antitumor and hemolytic activity relationships of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides. J. Peptide Res. 53:82–90 (1999).
Acknowledgements
Keywords
Red blood cells (RBC); hemolysis; hemolysin
Figure 1
Hemolysis of RBC by S. aureus laboratory strains PC1839 and AK3.
S. aureus strains, PC 1839 and AK3, at cell density of 107 cfu/ml, rupture the RBC within 5 min.
