Figure 1.

Protocol to stain PDX-ALL with CTV for subsequent MSC co-culture and flow cytometry analysis

(A) Schematic detailing the main concept of the CTV staining protocol described within this paper. An MSC co-culture was set-up, which was then co-cultured with PDX-ALL cells. Cells were stained with CTV and re-cultured on fresh MSC. PDX-ALL cells were subsequently treated with compounds as per experimental design. PDX-ALL cells were incubated for the desired time, cells were then harvested and passed through a 40 μM filter before flow cytometry analysis.

(B) Images depicting the various steps of the CTV labeling protocol. 10 million PDX-ALL cells were harvested from the MSC-PDX-ALL co-culture. Cells were centrifuged at 1500 RPM for 5 min. PDX-ALL cells were resuspended in 5 mL of 1× PBS and separately, resuspend 10 μL of CTV in 5 mL of 1× PBS. The PDX-ALL cells were combined with CTV solution, then incubated for 20 min. 1 mL of FBS was added before cells were incubated for 5 min. CTV-labeled cells were centrifuged and then resuspended in 20 mL of fresh SFEM media. The CTV-labeled PDX-ALL cells were then divided equally between experimental arms, drugs were added (if required) and then incubated for 5 days. PDX-ALL cells were harvested, filtered and 5 μL of propidium iodide was added before flow cytometry analysis.