PBMC Based Assays
Peripheral blood mononuclear cells (PBMC) provide selective responses to the immune system and are the major immune cells in the human body. PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. PBMCs based assays explore the role of these cells in immune modulation, cell proliferation/cytotoxicity and their role in targeting specific cancer cells.
The expert team at Cellomatics can support with PBMC assays within a variety of projects.
PBMCs – anti-CD3 stimulation
Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) were seeded onto anti-CD3 coated U-bottom plates and further stimulated with soluble anti-CD28 for 48 hours. PBMCs were also treated with Cyclosporin A prior to stimulation. Isotype of anti-CD3 was used as a background control. Supernatants were analysed for inflammatory markers using a Luminex Multiplex Assay (n=3±SEM; *p<0.05; ****p<0.0001).
PBMCs – anti-CD3/anti-CD28 stimulation
Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) were seeded onto anti-CD3 coated U-bottom plates and further stimulated with soluble anti-CD28 for 48 hours. PBMCs were also treated with Cyclosporin A prior to stimulation. Isotype of anti-CD3 was used as a background control. Supernatants were analysed for inflammatory markers using Luminex Multiplex Assay (n=3±SEM; *p<0.05; ****p<0.0001).
PBMCs – anti-CD3/anti-CD28 stimulation for 72 hours
Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) were seeded onto anti-CD3 coated U-bottom plates and further stimulated with soluble anti-CD28 for 24, 48 and 72 hours. PBMCs were also treated with Cyclosporin prior to stimulation. Isotype of anti-CD3 was used as a background control. Supernatants were analysed for inflammatory markers using Luminex Multiplex Assay (n=5±SEM; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).
PBMCs – anti-CD3 stimulation
Effect of ICAM-1 inhibitor on PBMC proliferation.
The absorbance (570 nM) for PHA stimulated PBMC cells was significantly higher when compared to the Vehicle Control (unstimulated) (##p<0.01). A statistically significant reduction of absorbance was observed for PBMC (PHA stimulated) treated with ICAM-1 inhibitor when compared to PHA stimulated control. Differences were identified using a one-way ANOVA followed by a Dunnett’s post-hoc multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ±SEM).
Effect of ICAM-1 inhibitor on release of inflammatory marker levels from PBMC.
PHA stimulation of PBMCs significantly increased the expression levels of inflammatory markers (e.g. TNFa) when compared to Vehicle Control (###p<0.001). A dose-dependent reduction in the release of the markers was observed for PBMCs treated with ICAM-1 inhibitor when compared to PHA stimulated PBMCs. Analysed using one-way ANOVA followed by Sidak’s post-hoc multiple comparison test (**p<0.01; ***p<0.001; ±SEM).
PBMCs proliferate in response to anti-CD3 stimulation
PBMCs from 2 different healthy volunteers were stimulated with increasing concentrations of anti-CD3 antibody (0 µM, 0.25 µM, 1 µM and 5 µM) over the period of 144 hours (6 days) and the cell proliferation was recorded. A dose dependent increase in PBMC cell counts was observed after 48 hours of stimulation (n=5±SEM).
PBMCs - LPS stimulation
Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) from 3 donors were stimulated with LPS for 24 hours in the presence or absence of Dexamethasone. Supernatants were analysed for inflammatory markers using Luminex Multiplex Assay. There was a significant increase in mediator expression with 100 ng/mL LPS, when compared to the unstimulated condition (***p<0.001). Significant differences were identified using a two-way ANOVA followed by a Šídák’s multiple comparisons test.
PBMCs - LPS stimulation
Freshly isolated Peripheral Blood Mononuclear cells (PBMCs) were seeded onto anti-CD3 coated U-bottom plates and further stimulated with LPS for 48 hours in the presence or absence of Dexamethasone. Supernatants were analysed for inflammatory markers using a Luminex Multiplex Assay (n=3±SEM; *p<0.05; ****p<0.0001).
PBMCs + MDP (muramyl dipeptide) stimulation
Levels of TNF alpha, IL1beta, IL-23 and IL-17 in PBMC supernatants of healthy volunteers following stimulation with MDP for 24 hrs in the absence or presence of p38MAPK inhibitor. Results are expressed in pg/mL.
PBMCs + MDP (muramyl dipeptide) stimulation
Relative mRNA levels of NOD1, NOD2, RIPK2, SLC15A4, NLRP1 and NLRP3 following stimulation of PBMCs (healthy volunteer) with MDP in the absence or presence of p38MAPK inhibitor.
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