Celltrace calcein red orange am

To better characterize the interactions between latex and cartilage particles, cartilage particulates were labeled with 20μM dichlorotriazinylaminofluorescein (DTAF, Sigma-Aldrich)^{28 (link)}. FLS were plated at either a low or high density overnight before treatment with 250 fluorescently labeled cartilage particles, green-yellow or unlabeled latex beads (Sigma-Aldrich) per cell for 2 days on glass coverslips. Prior to imaging, synovial cells were stained with CellTrace™ Calcein Red-Orange AM (Life Technologies) to identify if particles were internalized or in contact with the cell surface. Alternatively, cells were cultured and fixed in 4% PFA overnight, permeabilized with 0.1% Triton X (Sigma-Aldrich) and washed with PBS. After blocking with 1% BSA in PBS, cells were stained with TRITC-conjugated Phalloidin (Millipore-Sigma) and counterstained with DAPI. Z-stack images were taken and reconstructed using a Zeiss LSM700 Confocal microscope (Zeiss, Oberkochen, Germany). For actin-DAPI stained images with latex particles, non-fluorescent latex beads were used to eliminate the effects of channel bleed-through. For quantification of cell-particle interactions and spatial characterization, 3 blinded reviewers each counted 170 cells and 108 cells cultured with cartilage and latex particles, respectively.

Primary lung microvascular cells (175’000 cells) were plated on gelatin-coated μ-Slide I^0.2 Luer (ibidi GmbH) and grown to confluence for 2 days. The endothelial monolayer was manually perfused five times with 2x10⁵ of MC-38GFP cells in 100μl media. After 4 hours slides were perfused with CellTrace calcein-red-orange, AM (Life Technologies) stained bone marrow monocytes (2x10⁶ cells/ml) in HEPES/Ringer solution supplemented with 25% washed red blood cells. Using air pressure pump system (ibidi) we applied a flow rate of 2 dyne/cm². Mosaic images of slides were acquired using an inverted fluorescence microscope (Zeiss Axio Observer Z.1). Pictures were analyzed with ZEN software (Zeiss).