Why do I still see CD3⁺ T cells after differentiating M1 macrophages from PBMCs?

The most likely reason is an insufficient washing step during monocyte enrichment by adherence.
During macrophage differentiation from PBMCs, monocyte purification relies on adherence, while lymphocytes (including CD3⁺ T cells) should be removed during the washing steps.
 
Critical point in the protocol
The three washing steps described in the PromoCell protocol are essential. If washing is not performed thoroughly:

• Non‑adherent lymphocytes may remain
• These cells can persist and later be observed attached to or associated with macrophages

When performed correctly, the adherence‑based purification yields a monocyte population of >90% purity, which minimizes T‑cell carryover.