I am interested to calculate the total and differential hemocytes of lepidopteran insects. I am using a protocol prepared by myself in the lab, where the use of anti-coagulant buffer (98mM NaoH, 180mM NaCl, 17mM EDTA, 41mM Citric Acid, pH 4.5) is mentioned, but the hemocytes are coagulating. Kindly provide a suitable protocol or suggest how I can overcome the problem with procedure. Thanks in Advance.
Hi Rao, do you know if the hemocytes are sensitive to endotoxin (lipopolysaccharides)?
Steingrimur Stefanssson Sir, severalpapers shown the sensitivity of hemocytes to LPS, even my work is also correlated to study the endotoxin activity of invader nematodes with their symbiont bacteria on yemocytes profile
Hi Rao, endotoxin is everywhere and I suspect that you buffer has endotoxin.
If you can't get this buffer from a company that tests it for endotoxin, I would try making the buffer (and its components) using endotoxin-free water.
Hi Rao, you must keep cells and buffers always cold (4°C, work on ice). I wonder if ythe pH is to low? You can take a look in Willot et al. 1994, Immunochemical identification of insect hemocyte populations:
monoclonal antibodies distinguish four major hemocyte types in
Manduca sexta. AC Saline for M. sexta for example has a pH of 6.8. For determination of THC we normally dilute ten times with insect medium (Grace) and no coagulation will occur within the time needed to count.
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