Macsquant flow cytometry analyzer

To identify the MSCs’ predefined surface markers, 80% confluent G-MSCs were flowcytometrically analyzed [21 (link)]. The standard protocols for binding the primary antibodies and corresponding isotype controls were employed (Miltenyi Biotec). Afterwards, the results were assessed with a MACSQuant Flow Cytometry Analyzer (Miltenyi Biotec) and the analyzing software FlowJo 10 (Becton Dickinson).

The G-MSCs’ characterization using flowcytometry was performed before and after the TQ stimulation to determine changes in the expression of TLRs 1-10. For the staining of intracellular TLRs, the G-MSCs were initially fixed and permeabilized with a Fix & Perm kit (Cytofix/Cytoperm, BD Biosciences, Franklin Lakes, NJ, USA) before cell incubation. The following antibodies were used: anti-TLR1, anti-TLR3 and anti-TLR9 (all from Invitrogen, Waltham, MA, USA); anti-TLR2, anti-TLR4 and anti-TLR8 (all from Enzo Life Sciences, Lörrach, Germany); anti-TLR5 (R&D Systems, Hessen, Germany); anti-TLR6 (BD Life Sciences, East Rutherford, NJ, USA); and anti-TLR7 and anti-TLR10 (both from Invitrogen, Waltham, MA, USA). For the binding of the primary antibodies and their corresponding isotype controls, the standard protocols were followed, as described previously [1 (link),3 (link),9 (link)], and the results were then evaluated using the MACSQuant Flow Cytometry Analyzer (Miltenyi Biotec) and FlowJo 10 (Becton Dickinson, Franklin Lakes, NJ, USA).