Development of consensus flow-cytometry panels for use in the context of studies on immune system aging and CMV
Brighton, 3 October 2014
Dear Participants of the past ‘CMV and Immunosenescence’ Workshops!
With respect to the development of flow monitoring panels that we could all use in the context of CMV and ageing, we have completed a set of four whole blood T cell staining panels. These address the subsets that I believe were of greatest interest in discussions at the past two meetings in Cordoba and Parma.
We previously posted step 1 of this development, this is step 2.
The way these panels are designed, additional markers can be ‘dropped in’ in the Blue 2 channel (e.g. ECD conjugates). The AF700 CD45 can be replaced with another marker, e.g. an activation marker, if preferred. It was included for WBC threshold setting/dual platform absolute counting. Our LSR II has an Octagon on the violet laser, but we have only used Pacific Blue, BV 510, and BV 605 so panels can be used in standard configurations.
We had some difficulty with the CD8 APC Cy7 antibody, in particular when acquiring the next day. We tested various conditions that would allow us to use this reagent, however, eventually replaced it with a CD8 APC-H7 antibody as the conjugate disintegrated too quickly.
We are currently working on two remaining panels, one for B cells and one for antigen-presenting cells. The four T cell panels have been included in our current studies. About 35 samples have been run with the T cell and the Treg panel in CMV+ and CMV- people (about 50% each) and sample FCS files will be posted here shortly. You can now also find the exact protocol and the reagents that were used including the exact amounts of each antibody as per our extensive titrations in the beginning. I am happy to share the results from titrations with anyone who is interested.
Many thanks to Dr. Stefano Caserta, Dr. Nadia Terrazzini, and Mr. George Morrow who have spent many hours on getting these panels right!
I am looking forward to your feedback and hope to be able to discuss in more detail with you soon in Amsterdam!
Whole Blood Panels CMV/ageing - Laboratory Protocol (Brighton Immunology Lab):
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