**3.5 Co-culture of MSCs with cancer cell line**

There are various 2-D or 3-D, dyeing or not dyeing, co-culture models of human MSCs and other cell sources to study cell–cell interaction, cell proliferation, MSCs' immunomodulatory capacity, and the cellular contribution of each cell type. These methods making co-cultures of MSCs and other cells are well-established, such as co-cultures of MSCs and human peripheral blood mononuclear cells [33, 34], MSCs and T cells [35], MSCs and human hematopoietic stem cells [36], MSCs and umbilical vein endothelial cells [37]. MSC-cancer cell (PC-9) co-culture will be described in this section (**Figure 3**).


#### **Figure 3.**

*Human BM-MSCs-PC-9 co-culture. Representative images ((A) 5×; (B) 20×) of co-cultures of human BM-MSCs labeled with CellTracker™ red dye and PC-9 cells transfected with copGFP, nuclei counterstained with DAPI.*

*<sup>3.5.1</sup> Preparation of MSCs with CellTracker™ Red dye (CellTracker™ CM-DiI, C7000)*

	- 1.PC-9 cells labeling with copGFP (Santa Cruz, sc-108083) is performed using the lentiviral technique. Lentivirus is produced by using Lipofectamine™ 2000 (Invitrogen), according to the manufacturer's instructions. After transfection, PC-9 cells expressing copGFP may be isolated via puromycin (2 μg/mL) selection.
	- 2.Continue to serially passage.
	- 3.Collect PC-9 cells for cell co-culture.
