Table 4.

Troubleshooting table.

Step	Problems	Causes	Suggestions
1.2, 1.3	Excessive cell death	Tissue left at room temperature Enzymatic dissociation for extended periods	Always keep tissue on ice Enzymatic dissociation should be performed only for 10 min
2.4	No extracellular staining	Inappropriate antibody dilution Inadequate incubation time	Use recommended dilution of antibody Use recommended incubation conditions
3.2	No intracellular staining	Intracellular staining performed using FACS buffer	Permeabilization buffer should be used for intracellular staining
3.7	Spillover of fluorophores	Flow cytometry instrument not setup appropriately	Run recommended Cytometry Setup and Tracking (CST) beads Run compensation controls
4.1	No tetramer staining	Mis-match MHC haplotype	Refer to mouse alloantigen database [21]