When using less than 25 Moi of virus sensitivity makes the assay unreliable.
The 96-well and 384-well results shown in Figure 4 are not comparable in their signal height as the 384 well plates were lysed in 2.5 fold more lysis buffer hence the relative luciferase units are approximately 2.5 fold lower than those obtained using 96 well plates. More lysis buffer was added to 384 well plates to ensure there was enough lysate to perform luciferase assay.
The FLUOstar Omega plate reader gives the potential to analyse multiple cytokines on the same assay plate when using the grouping feature. This pilot study shows that the human primary macrophages can be cultured in 384-well plates without any changes in morphology or cytokine expression.
Reducing the number of cells needed for each assay without altering the sensitivity will now open possibilities to do screening assays on primary cells to look for new targets in autoimmune diseases. Previously this has only been possible in cell lines that do not respond to TLR ligands in the same way as primary cells. Successful Adenovirus infection of primary human macrophages in 384-well plates also allows reporter assays to be performed.
During the autoimmune disease Rheumathoid Arthritis immune competent cells such as macrophages and T-cells accumulate in joints. There they secrete the cytokines IL6, IL10 and TNFα. These activate the transcription factor NFκB which promotes inflammation.
Using the FLUOstar® Omega multi-mode plate reader cytokine secretion and NFκB-induction were measured in macrophages upon various stimuli. The cytokines IL6 and TNFα were quantified in the culture supernatant by absorbance-based ELISAs. A luciferase reporter assay was used to detect transcriptional activation of NFκB. Lipopolysaccharide stimulated macrophages showed a higher luminescence, indicative of NFκB activation, than their non-stimulated controls.
The FLUOstar® Omega analyses multiple cytokines on the same plate. We show that human primary macrophages can be cultured in 384-well plates without any changes in morphology or cytokine expression. Reducing the number of cells for each assay without altering the sensitivity now opens possibilities to do screening assays on primary cells to look for new targets in autoimmune diseases.