Table 8:

Trouble Shooting

Issue	Possible Cause	Action
No cells in capture site	Possibly caused by clogs in inlet, too few cells loaded, cells too small for chip size.	If no clog is visible and all reagents were properly loaded then rerun capture cycle up to 2 times after carefully recalculating cell concentration. Recalculate cell size to ensure correct IFC is being used.
Too many cells in capture site, clumps	Loading too many cells, loading cells too large for selected chip, incomplete digestion of sample.	Reduce cell concentration for future experiments with given cell type. Adjust cell isolation protocol to minimize clumps. Select size appropriate IFC. Use FACs to separate to pre-isolate cells of appropriate size.
No liquid in outlet wells of IFC	Clog, failure to properly prime IFC, failure to add harvest reagent to all appropriate wells, bubbles in IFC loading wells.	If clumps observed, adjust cell isolation protocol. At any step before loading IFC into Fluidigm C1 machine carefully check each well that reagents were added to ensure that they are appropriately loaded and that no bubbles are present at the bottom of the wells.
No cDNA detected after IFC amplification for individual sample(s)	Poor cell viability, presence of debris, Rnase contamination, low RNA content of cell	Check viability of cells and revise isolation protocol if necessary to maximize viability while reducing debris. FACs, Density separation, or other sorting may be done to remove debris. Ensure work area is clean with Rnase degrading chemicals.
No cDNA detected after IFC amplification in all samples	Clog, reagent missing mixture or not loaded into IFC, loss of cell viability during preparation.	Check for clog, cell viability, that cells are of the appropriate size for IFC, and that each well is appropriately loaded and that no bubbles are present at the bottom of the wells.