Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.
Please check your plastic cell culture dishes, as monocytes are known to get intrinsically activated by adherence to plastic surfaces. For more details, please see the attached publications:
Do you use heat inactivated FBS or FCS. If you are not using heat inactivated FCS or FBS there could be a background activation due to presence of complement receptors.