Table 3. Troubleshooting.

Problem	Possible Cause	Solution
Basic Protocol 1: Cells are erroneously injected onto (rather than into) the pancreas. This will lead to cancer cell dissemination into the abdominal cavity.	Needle positioned incorrectly (piercing through the tissue instead of remaining within it).	The entire organ should be immediately flushed with sterile water using a 1-ml syringe equipped with a wide bore (22G to 24G) needle. This will result in death of the cancer cells by osmotic shock. A second injection can be attempted after the flush.
Basic Protocol 1: Bleeding	Damage to local blood vessels.	Use a cotton swab to apply gentle pressure on the source of bleeding. Upon achievement of hemostasis, the procedure can continue.
Basic Protocol 2: The contents of the 50-ml tube do not elute through the strainer.	The solution is highly cellular (high cell density in the digested tissue or large size of the tumor/liver).	Use a 1,000-μl pipette tip to stir the solution, and move the larger, undigested bits of tissue towards the side of the strainer. If necessary, further dilute the solution by adding more R10.
Basic Protocol 2: The cell suspension from the digested liver and/or tumor and Ficoll admix during centrifugation.	The acceleration and/or deceleration rates were not set to 0, disrupting the formation of layers.	Immediately stop the centrifuge run, mix the sample by gently inverting the tube a few times and layer over Ficoll again, ensuring the acceleration and deceleration rates are both set to 0.
Basic Protocol 3: The sample viability observed on the flow cytometry analysis software is considerably higher than that recorded during manual cell count in Basic Protocol 2.	Cells were incubated with the viability dye in a protein-rich medium, causing the amine-reactive viability dye to bind non-specifically to the proteins in the medium and reducing the amount of dye available to bind to dead cells.	Repeat staining using DPBS in the viability dye incubation step.
Basic Protocol 3: The sample viability observed on the flow cytometry analysis software is considerably lower than that recorded during manual cell count in Basic Protocol 2.	Cell loss during handling and staining or excessive fixation.	Repeat staining ensuring: Adherence to the manufacturer’s instructions for the use of the viability dye and fixation/permeabilization reagent. Titration of the viability dye. Appropriate cell handling (e.g., keeping cells on ice).
Basic Protocol 3: Unmixing errors are observed (e.g., hypernegative populations or oddly-shaped negative populations).	Reference controls of inadequate quality were used during unmixing.	Repeat unmixing with autofluorescence extraction, ensuring: The unstained controls are from the same anatomical district as the multi-color experimental samples. The mean fluorescent intensity (MFI) of the positive population in the single-stained controls is greater than or equal to that of the positive population in the multi-color experimental samples. All controls are treated in exactly the same way as experimental samples (e.g., same duration of fixation and incubation with antibodies).