I am isolating bone marrow neutrophil from C57b mice using the percoll method (62.5%)
These neutrophil are labelled with calcienAM for 30min, then added to a boyden chamber for chemotaxis towards varying concentrations of KC (CXCL1)
The KC is in lower chamber, cells at 1x10^6/ml in upper chamber and migrate across a fibronectin coated membrane.
However, the cells do not migrate towards KC, and it seems to actually prevent migration at 20-100ng/ml.
The assay is carried out in RPMI (without phenol red) in the absence of FBS.
Can anyone speculate why the cells would not migrate to KC??
This is supposed to be the positive control in my experiment and holding me up considerably
Thank you in advance