Table 1.
Antibody composition of staining master mix (basic panel and Treg panel) applied for multicolor flow cytometry.
Figure 1.
The effects of overnight shipping on PBMC yield and survival.
The cell number of blood derived PBMC was determined based on Trypan blue exclusion. A) Blood was collected in S-Monovette (Sarsted) or K2E Vacutainer (BD) tubes from 4 donors on site or at another location. Shipped samples were transported in the collection tube. PBMC yield was calculated per volume (106 living PBMC/ml blood). B) Yield (106 living PBMC/ml blood) is shown for PBMC derived from non-shipped vs. shipped blood (n = 9) prior to freezing. C) Viability (% of living cells/total cell count) is shown for non-shipped vs. shipped samples (n = 9), prior to freezing. D) Post-cryopreservation recovery is calculated for thawed samples (non-shipped vs. shipped, n = 9) and is provided as percentage of living cells from 107 total (frozen amount/vial).
Figure 2.
The effects of overnight shipping on lymphocyte subpopulations.
PBMC were analyzed post cryo preservation by flow cytometry, to determine the ex vivo frequencies of CD3+CD4+ T cell, CD3+CD8+ T cell, CD3+CD127−CD25+CD4+ regulatory T cell, CD3−CD19+ B cell and CD3− CD56+ NK cell populations. A) The gating strategy is presented on dot plots from one representative donor. Abbreviations: EM-effector memory cells, CM-central memory cells B) Bar graphs indicate the average frequencies for all tested donors (n = 9) in non-shipped vs. shipped samples. All graphs are shown with SEM values. Before-after graphs show the same data broken down for each individual.
Figure 3.
The effects of overnight shipping on T cell proliferation.
PBMC from non-shipped vs. shipped samples were thawed, loaded with eFluor670 cell proliferation dye and stimulated with plate bound anti-CD3 (OKT3) and soluble anti-CD28 antibodies for 6 days (as described in the methods). Cells were harvested and proliferation was analyzed by flow cytometry. A) A representative graph is shown for CD8+ and CD4+ T cell proliferation. B) Bar graphs summarize the percentage of proliferating CD8+ (left) and CD4+ (right) T cells of all donors (n = 9).
Figure 4.
The effects of overnight shipping on cytokine production.
PBMC were thawed and in vitro stimulated (anti-CD3 + anti-CD28) for six days. Supernatants were collected from the plate-wells and cytokine levels were determined with cytokine bead array for all donors (n = 9). Bar graphs show unstimulated and stimulated conditions both for non-shipped and shipped samples. All graphs are shown with SEM values. Before-after graphs visualize the cytokine levels on individual level. A) Th1/Th2 cytokines; B) Th17 cytokines.
Figure 5.
The effect of overnight shipment on signal transduction machinery.
PBMC were stimulated with anti-CD3 (OKT3) + anti-CD28 antibodies for 5 min (A) or with rhIL-6 for 30 min (B) at 37°C in RPMI medium and lysed immediately after stimulation. Post-nuclear lysates were subjected to SDS-PAGE and proteins were transferred onto nitrocellulose membrane. Subsequently, membranes were probed with anti-pERK (A) or with anti-p-STAT3 (Y705) (B) antibody. Equal protein loading was controlled by probing the membranes against β-actin. Results are shown in a form of scanned blots for 2 representative donors out of 4 tested individuals (for both signaling pathways).