Hi,
I am trying to isolate eosinophils from human whole blood for RNA sequencing, which requires a high purity. I also wanted to use a method which manipulates the cells as little as eosinophils can become activated during isolation i.e. using density centrifugation.
I have tried using the direct from whole blood eosinophil isolation kit from StemCell, however, there still seems to be some contaminating populations afterwards (populations in red circles in attached figure, which shows isolated eosinophils using the StemCell kit from a healthy donor). These populations were also seen in the TDS they supply and was seen when the Miltenyi Biotec Direct from whole blood kit was used.
LARGE POPULATION: I have tried staining these populations to try and identify the cells, but they are negative for CD16, CD123, CD303, Siglec-8, Lineage cocktail, HLA-DR and FceR1a. I have tried two different CD16 clones for the population that we expected to be neutrophils (larger population), but they were still negative.
SMALL POPULATION: Some cells in the smaller population have stained positive for CD34, HLA-DR, CD294 and CD193. Therefore, I used a CD34-biotin antibody to try and remove some cells and while this has been partially successful for the smaller population, there are still some cells left.
I have also tried an extra isolation step in the magnet with more Rapidsphere magnetic beads, but there was no difference.
Any suggestions, experience or guidance would be very much appreciated.
Rebecca
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