I'm using the protocol linked below, which calls for blood collection into an EDTA laced vacutainer. I'm waiting on arrival of EDTA vacutainers, but I have heparinised ones laying around and I'm wondering if they would be equally suitable for blood collection prior to neutrophil isolation. After an initial centrifugation, this protocol calls for dilution of cells in a PBS buffer containing very low amounts of EDTA (0.2ug/ml).
I'm collecting sheep blood (~10ml) and plan to use isolated neutrophils several hours later for a sperm binding assay.
http://download.springer.com/static/pdf/501/chp%253A10.1007%252F978-1-59745-467-4_3.pdf?originUrl=http%3A%2F%2Flink.springer.com%2Fprotocol%2F10.1007%2F978-1-59745-467-4_3&token2=exp=1439963363~acl=%2Fstatic%2Fpdf%2F501%2Fchp%25253A10.1007%25252F978-1-59745-467-4_3.pdf%3ForiginUrl%3Dhttp%253A%252F%252Flink.springer.com%252Fprotocol%252F10.1007%252F978-1-59745-467-4_3*~hmac=a1078c6170f942c98e1e76d986de2b2141a8e6c5c8040c554691af3e4ebbaa02
Taylor,
For many purposes, either anti-coagulant would be fine. However, for an adhesion study such as yours, heparin may be a problem. Heparin will bind to the PMN and can interfere with adherence even after washing it out. This is has been shown for HUVEC binding among others:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1571874/
Also, isolated PMN must be stored cation free and on ice to minimize cell damage. Nonetheless, isolated PMN will degrade fairly quickly in storage, so the sooner they are used, the better. Several hours of storage is approaching their limit. If you have a choice, it's preferable to store the whole EDTA treated blood rather than the purified PMN. That may be your plan already.
Using a link to give details of the isolation method was a good idea. However, it required paid access, so I didn't look at it.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1571874/
Yes ! I've used both anticoagulants for neutrophil isolation and they worked fine.
Heparin activates neutrophils. EDTA or Citrate might be better. Try to measure the activity of neutrophils in the blood ROS Generation Assay (BRGA) where Isolation of them is not necessary.
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