Table 11.

Troubleshooting Guide for Bone Stromal Cell Staining and Cytometry

Problem	Possible Cause	Solution
Low Signal/ High noise	Contamination with B or T cells	Gate or sort B and T cells out of the target population by staining samples with PE/Cy7 or PE/Cy5 conjugated antibodies to CD3, CD4, CD19, and CD45 (McLaughlin et al., 2008; Perfetto et al., 2004; Baumgarth and Roederer, 2000). In this case, the cells can be removed from the analysis by gating allowing for a higher signal to noise ratio. Depending upon the cytometer, other fluorophores can be used to create a “dump” channel where non-target populations can be measured or sorted. Additional markers for B and T cells can also be included.
Low Cell Isolation	Insufficient collagenase digestion	Try incubating in collagenase I for longer times or at a higher concentration. Another potential issue is too small vessel size for the tissue pieces to move freely. Tissues can also be cut smaller to increase the surface area exposed to the collagenase.
High Autofluorescence	Fixation method	Autofluorescence can occur with formaldehyde fixation for hematopoietic lineage cells. Use another fixative to reduce autofluorescence.
Loss of Fluorescence Signal	Fixation duration	Fixation at 1% formaldehyde should not affect fluorescence signal (including for PE and APC) if samples are run within two weeks. If longer storage is required or if the signal degrades, fixation can be performed overnight, followed by centrifugation and resuspension in PBS for longer storage.