Strains were inoculated into 2mL of TSB from blood agar plates and incubated at 37°C for 6h. Cultures were then diluted 1:1000 in 10mL of TSB and incubated for 16-18h at 37°C, 200rpm. Cells were then pelleted by centrifugation at 3500g for 10 min. 1.5mL of supernatant was aliquoted and stored at -20°C until further use. A sheep erythrocyte solution was prepared by diluting a 50% blood suspension in Alsever-buffer (Labor Merck, Würzburg, Germany) 1:25 in PBS to a final concentration of 2%. 100μl of the erythrocyte solution and 100μl of culture supernatant were then mixed and incubated at 37°C for one hour under gentle agitation. As internal reference the erythrocyte solution was mixed with pure TSB and as positive control the solution was mixed with TSB+1%Tween. After one hour of incubation, samples were centrifuged at 13 000rpm in a table centrifuge and 150μl of supernatant were transferred to a 96-well flat-bottom plate (Greiner Bio-One, Kremsmünster, Austria). Absorption at 541nm as a measure of free haemoglobin in the supernatant was read on an ELISA reader (Tecan infinite M200, Männedorf, Switzerland). Absorption was then normalized to absorption of the internal reference. Experiments were conducted in triplicates. Biological replicates were averaged for statistical analysis.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.