I am trying to isolate neutrophils from bovine and rat whole blood samples. I have purchased the Dynabeads Epoxy and antibody linking kit and have a bovine CD44 and rat NCF2 antibody that can be linked to the beads. Although I can easily find a protocol online about how to link the antibody to the beads, when it comes to working with my whole blood sample to isolate the neutrophils I am having some difficulty determining the amount of whole blood sample that would be appropriate per mg of beads. I know that this might greatly depend on the affinity of my antibody, however I am just beginning to develop and test the protocol so I would really appreciate some general guidance.
Thank you so much for your help.
We have never used whole blood for cell separation. I think that it is better at first to isolate all blood cells, to remove erythrocytes and than to isolate neutrophils. If you will know the cell number and the ~ percent of neutrophils it would be possible to calculate the bead/cell ratio.
Good luck!
Hi
Most depletions and positive isolations can use whole blood as a starting sample. When using whole blood, add washed Dynabeads directly to the sample. Use 4-10 Dynabeads per target cell. Keep the Dynabeads concentration 1 x 107 beads / ml sample for positive isolation and 2 x 107 beads / ml sample for depletion. It is possible to use diluted blood when the sample is very viscous - dilute whole blood in equal volume of cold PBS immediately before adding Dynabeads.
Washed Blood
The yield of some cells such as monocytes can be significantly increased by washing the sample before adding the Dynabeads. CELLection products also require washing if whole blood is used as a starting sample. Sufficient washing is achieved by centrifugation and removal of the blood plasma followed by a single wash in PBS /0.1% BSA/0.6% Na citrate. After washing, the sample is re-suspended in PBS /0.1% BSA/0.6% Na citrate to the original volume. 0.6% sodium citrate is added to counteract microagglutination/cell aggregation.
kind regards
ketil
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