Phospho flow is a powerful approach to detect cell signaling aberrations, identify biomarkers and assess pharmacodynamics, and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here, B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions, fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281576PMC
http://dx.doi.org/10.1038/s41598-018-36121-9DOI Listing

Publication Analysis

Top Keywords

phospho flow
20
signaling responses
8
cryopreserved samples
8
reproducibility phospho
8
phospho
5
flow
5
cryopreservation primary
4
cells
4
primary cells
4
cells minimally
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!