When comparing plots of cells live vs post banking with PROT1 (or Smart Tubes), you will see lower FSC/SSC in the sample post banking. Increase FSC and SSC voltage to keep the lymphocytes high enough to gate, but even with higher voltages you will not get exactly the same FSC/SSC that you see in live samples. There's much that stays the same: debris will still have smaller FSC/SSC than lymphocytes and lymphocytes will have lower FSC/SSC than monocytes and grans, Just be aware that there won't be as much separation as before and there can be more overlap than with the live sample (ie. degranulated grans can have FSC/SSC that badly overlaps with other populations).
If you are working with freshly collected whole blood from a healthy donor there probably aren't any dead or dying cells in your sample, but since live/dead discrimination often comes up when discussing scatter I thought I might mention the following:
If you think you have any dead or dying cells it may be advantageous to stain with cleaved PARP:
cleaved PARP
Epitope: D214
Clone: F21-852 (BD Biosciences is one supplier of this clone)
Publications that used this antibody with Smart Tube stabilized samples include:
Single-cell mass cytometry adapted to measurements of the cell cycle
Cytometry Part A
Gregory K. Behbehani, Sean C. Bendall, Matthew R. Clutter, Wendy J. Fantl, Garry P. Nolan
and:
Mass cytometric functional profiling of acute myeloid leukemia defines cell cycle and immunophenotypic properties that correlate with known responses to therapy
Cancer Discovery, 2015
Behbehani et al
and methods paper:
Cell Cycle Analysis by Mass Cytometry.
Methods Mol Biol. 2018;1686:105-124. doi: 10.1007/978-1-4939-7371-2_8
Gregory K. Behbehani
You can view our publications page on our website : https://www.smarttubeinc.com/publications.htm
0 Comments