Figure 1. Flow Cytometry Analysis of Cultured Human Peripheral Blood Mononuclear Cells (PBMCs) with Gating on Viable CD4⁺ and CD8⁺ T Cells

Figure 2. Flow Cytometry Analysis of Fresh Human Regulatory T Cells in Total PBMC Sample with Gating on the CD45⁺ CD3⁺ CD4⁺ CD25⁺ and FOXP3⁺ Population

Figure 3. Human PBMC Proliferation After Activation

Human peripheral blood mononuclear cells (PBMCs) were stimulated with different concentrations of ImmunoCult™ Human CD3/CD28 T Cell Activator (Catalog # 10971) for 5 days. PBMCs were labelled with cell tracking dye, and viability was measured by gating on 7-AAD (viability dye) negative cells. Cell division and proliferation were measured by flow cytometric analysis. Each peak represents a cell division.

Figure 4. Intracellular Flow Cytometric Analysis of Immune Cytokines

Activated human T cells (gated on CD4⁺ and CD8⁺ populations) were analyzed by intracellular flow cytometry for IFN-γ and TNF-α expression. After the cells were fixed and permeabilized, antibodies against the cytokine of interest were added to the cell suspension, followed by flow cytometry.

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Figure 5. Meso Scale Discovery Multiplex Platform

Schematic representation of Meso Scale Discovery multiplex platform. STEMCELL’s Contract Assay Services (CAS) group is a certified CRO partner of Meso Scale Discovery.

Figure 6. Compounds Can Be Screened for Immunomodulatory Effects Using the Treg Cell Suppression Assay

Fresh Treg cells and PBMCs were purified from a healthy donor. Treg cells and PBMCs were co-cultured in the presence of a solvent, an immunomodulatory compound, or a negative control article and then activated for 4 days with ImmunoCult™ Human CD3/CD28 T Cell Activator in ImmunoCult™-XF T Cell Expansion Medium. (A) The suppression response at the 1:2 Treg:PBMC ratio was 45% for the solvent control, 37% for the negative control article and 89% for the immunomodulator. Co-culturing with conventional CD4⁺CD25^- T cells showed < 10% of the suppression response (mean ± SD, n = 3, single donor). (B) Control experiments demonstrating the proliferation of responder cells in the absence of Treg cells are shown. Cells were cultured in conditions as described above. The immunomodulator alone had a minimal effect on responder cell proliferation (mean ± SD, n = 3, a representative single donor).

Figure 1. Mobilization of Hematopoietic Progenitors Induced by rhG-CSF and AMD3100

C3H/HeN mice were treated with 5 µg rhG-CSF per day on days 0 - 2. Sixteen hours after the last G-CSF injection, one group of 5 mice was treated with 5 mg/mL of AMD3100 (also know as Mozobil or Plerixafor). Ninety minutes after treatment with AMD3100, mice were sacrificed and peripheral blood (PB) was collected. The progenitor content in the PB of each mouse was then assessed using the CFU assay. Mice treated with rhG-CSF alone showed a 10-fold increase in the number of CFUs per mL of PB, and mice treated with rhG-CSF and AMD3100 showed a 50-fold increase in the number of CFUs per mL of PB, when compared with vehicle control.

References

1. Szilvassy SJ et al. (2002) Quantitation of Murine and Human Hematopoietic Stem Cells by Limiting-Dilution Analysis in Competitively Repopulated Hosts. In: Klug CA & Jordan CT (Eds.), Methods in Molecular Medicine: Hematopoietic Stem Cell Protocols (pp. 167-187). Totowa, NJ: Humana Press.
2. Miller CL & Eaves CJ. (2002) Long-Term Culture-Initiating Cell Assays for Human and Murine Cultures. In: Klug CA & Jordon CT (Eds.), Methods in Molecular Medicine: Hematopoietic Stem Cell Protocols (pp. 123-141). Totowa, NJ: Humana Press.