HTFC screening protocol

To develop a ?no-touch? antibody staining method to be able to perform flow cytometry on adherent cells in a high-throughput fashion. Conclusion: We show that we have generated a universal ‘no-touch’ antibody staining method to perform HTFC. The unmodified protocol works with several cell lines from different cellular origins. We show that multiplexing the staining does not influence the staining efficacy. The obtained results are in concordance with conventional FACS. Fluorescent labelled beads (CS&T beads, Becton Dickinson) were used to check the performance and verify optical path and stream flow of the flow cytometer. This procedure enables controlled standardized results and allows the determination of long-term drifts and incidental changes within the flow cytometer. No changes were observed which could affect the results. In every experiment, appropriate controls have been taken into account: unstained, medium controls. Every condition, with every staining combination and every cell line has been performed at least in duplicate within individual experiments. Effects of TNF-α/IFN-γ are confirmed with ‘conventional’ flow cytometry.