Journal: bioRxiv

Article Title: Label-free lymphocytes reconstitution using side scatter for optimal T cell manufacturing

doi: 10.1101/2020.11.09.375584

Figure Lengend Snippet: ( A-C ) Expansion efficiency of lymphocytes derived from SSC hi , SSC low , or reconstituted SSC hi +low cells at a ratio of 1:1 are represented by TNC fold increase ( A ), CD3 + CD4 + T ( B ) and CD3 + CD8 + T fold increase ( C ). Data are presented as means ± S.E.M (n = 3). Paired two-tailed t -test between SSC low and SSC hi group was performed. Error bars indicate mean ± SEM. ( D and E ) Compositional change of naive/memory subsets in CD3 + CD4 + T ( D ) or CD3 + CD8 + T cells ( E ) plotted along the days of expansion. Error bars indicate mean ± SEM. ( F ) Percentage and relative fluorescence intensity (RFI) of CD27. ( G-I ) Representative histogram plot of IFN-γ/IL-2/TNF-α expression of cell product at day 11 post expansion derived from cell groups with different side-scattering intensity ( G ). Multifunctional cytokine expression of total CD4 + T ( H ) or CD8 + T ( I ) cells at day 7 and day 11 post expansion (n = 3) with 2 independent experiments for donor 1 and 2. Migration capacity of T cells at day 11 post expansion derived from SSC hi , SSC low and SSC hi +low groups. Error bars indicate mean ± SEM. Responsiveness of effector cells descended from SSC hi , SSC low and SSC hi +low groups at day 11 post expansion to multi-stimulation by CD3/CD28 engagement with IL-2 ( top ) or homeostatic cytokines IL-7 plus IL-15 ( bottom ). Duplicates for each sample were performed. Unpaired two-tailed t -test between SSC low and SSC hi group was performed. Error bars indicate mean ± SEM. ( L and M ) All phenotypic parameters and cytokine-expressing values were integrated together for PCA calculation. For data from day 7, IFN-γ + IL-2 + / IFN-γ + TNF-α + / IFN-γ + IL-2 + TNF-α + were combined as “IFN+Multi”. “H” and “L” indicate “SSC hi ” and “SSC low ”, respectively. See also Figures S4A-4C and S4E-4G for individual data, and Figure S4D for gating method in cell differential lineages characterization.

Article Snippet: Antibodies for intracellular staining include granzyme B-PE (BD Biosciences, GB11; cat #561142), IFN-γ-FITC (Miltenyi Biotec, cat #130-090-433), IL-2-PE (Miltenyi Biotec, cat #130-090-487) and TNF-α-APC (Miltenyi Biotec, cat #130-091-267).

Techniques: Derivative Assay, Two Tailed Test, Fluorescence, Expressing, Migration

Journal: PLoS ONE

Article Title: Dynorphin 1-17 and Its N-Terminal Biotransformation Fragments Modulate Lipopolysaccharide-Stimulated Nuclear Factor-kappa B Nuclear Translocation, Interleukin-1beta and Tumor Necrosis Factor-alpha in Differentiated THP-1 Cells

doi: 10.1371/journal.pone.0153005

Figure Lengend Snippet: The LPS-stimulated THP-1 cells were treated with the N-terminal fragments of DYN 1–17 and U50,488H at 0.1 μM, 1 nM and 10 pM for 24 hr. The culture supernatants were collected and IL-1β and TNF-α release was measured using IL-1β and TNF-α AlphaLISA kit, respectively. The AlphaLISA signal was read using an Enspire-Alpha 2390 Multilabel Plate Reader. Non-stimulated THP-1 cells (NS) served as negative control. The release of IL-1β and TNF-α in each treatment group was normalised and expressed relative to LPS-stimulated control group. Data shown are the means ± S.E.M. of at least three independent experiments performed in triplicates. Statistical significance as denoted by * and # represent the IL-1β and TNF-α release between peptide-treated (solid-line) and LPS-stimulated control group or ML-190-treated group (dotted-line), respectively, p ≤ 0.05.

Article Snippet: The level of human IL-1β and TNF-α secretion was quantified using AlphaLISA kit (Perkin Elmer, VIC, Australia) according to the manufacturer’s protocol.

Techniques: Negative Control, Control