Computational protocol: Roles of calpain-calpastatin system (CCS) in human T cell activation

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Protocol publication

[…] For the majority of experiments described above it was performed with FACSVerse™ flow cytometer, while acquisition of data concerning the detection of phosphorylated signal molecules (phos-flow) - with FACS Aria III™ cytometer, and with their respective on-board acquisition software (both Becton Dickinson, USA). At least 3*104 cells were acquired from each ex vivo sample and at least 5*104 cells were acquired from each sample of PBMC cultured for 72 or 120 hours. Resulting raw data were analyzed using the FlowJo™ and Flowing Software 2.5.1 (© Perttu Terho, Turku University) analysis programs. Analysis of changes in the amounts of phosphoproteins was performed with the help of cloud-based Cytobank™ platform. At analysis, the lymphocytes were first identified by forward and side scatter gating and then by CD3, CD4, and CD8 expression, respectively (with further identification of T cell subpopulation according to the staining scheme used); finally, the signals from channels corresponding to the molecule(s) of interest (CCS, cleaved tBOC, and phosphoproteins) were acquired for the subpopulations defined as above. [...] Statistical analyses were performed with STATISTICA 8™ (StatSoft, Poland) using the unpaired or paired Student T test or non-parametric Mann-Whitney U-test where applicable. All values are shown in the graphs either as scatter plots with marked means +/− standard deviation (SD), or as bars corresponding to means with marked SD […]

Pipeline specifications

Software tools FlowJo, Flowing Software, Statistica
Databases Cytobank
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens
Diseases Mastocytosis, Systemic