Hi everyone, I have been trying to isolate neutrophils from mouse blood with lower cell counts, around 1.2–1.5 × 10⁵ cells/mL. I wonder if anyone can help me trouble-shoot?.
I tried using BD Lysis Buffer for erythrocytes lysis, followed by centrifugation at RT (300g) before performing magnetic selection of neutrophils. I also perfomed all the lysis steps on ice to minimize any temperature-dependent variation in lysis. I strictly followed the Miltenyi protocol for isolation, keeping every step cold. However, I have not achieved better cell counts so far.
Hi Ragini, Please pay aatention that the hair of mice can cause hemolysis. When collecting blood by enucleation, the whiskers of the mice should be trimmed first. Then, let the blood droplets fall into the centrifuge tube to avoid contact with the hair or gloves, which may lead to hemolysis.
Feel free to reach out for more details.
Hope this helpful.
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