Falcon hts fluoroblok inserts

Migration assay was performed in accordance with the Boyden chamber method, using cell culture inserts with a light-opaque polyethylene terephthalate 8-μm microporous membrane (BD Falcon HTS FluoroBlok inserts; BD Biosciences). DPCs were dissociated with accutase, counted, and incubated with 5 μg/mL of calcein AM (BD Biosciences) for 15 minutes at 37°C and 5% CO₂. Cells were washed with PBS, centrifuged, and resuspended with serum-starved (1% FBS) medium. Cells were counted one more time, seeded in the upper chamber (cell insert), and incubated for 24 hours. Subsequently, cells were fixed with 4% PFA, washed with PBS, and observed under the microscope. The total number of migrated cells observed in the bottom of the chamber was counted in four different pictures taken per chamber/insert.

U937 cells were purchased from the American Type Cell Collection. The cells at 2–3×10⁶ cells/ml were grown in RPMI 1640 medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO). Fresh U937 cells were then incubated with 10 µM Calcein AM (BD Biosciences) at 37°C for 1 h with 5% humidified CO₂. Subsequently, an aliquot of U937 cells (∼1×10⁶ cells/ml) suspended in serum-free RPMI 1640 medium was added to the upper compartment of the 24-well BD Falcon HTS FluoroBlok Inserts (BD Biosciences) [25] (link). This apparatus has a polyethylene terephthalate (PET) membrane (8 µm pore size) that blocks the transmission of light from 490 to 700 nm. This allows detection of cells present in the lower compartment only. The cells were allowed to migrate into the lower compartment at 37°C for 2 h in the presence of _pE-MCP1-6His, with the recombinant _Q-MCP1 (PeproTech) as a negative control. Once cells migrate through the pores of the PET membrane, they are no longer shielded from the light and can be detected by a fluorescence plate reader (Bio-Tek-Synergy HT Microplate Reader, Bio-Tek Instruments). Chemotactic index (CI) was calculated from the cell migration activity towards chemoattractant divided by the migration activity in the absence of chemoattractant. The CI values, shown as mean ± SEM, were calculated from five independent experiments.

Migration assay was performed according to the Boyden chamber method, using cell culture inserts with a light-opaque, polyethylene terephthalate, 8-μm, microporous membrane (BD Falcon HTS FluoroBlok inserts, BD Biosciences). BMSCs were dissociated with accutase, resuspended in basal medium and counted. Cells were then washed with PBS, centrifuged, and resuspended with serum-starved (1% FBS) medium. Cells were counted one more time, and 5000 cells contained in 250 μL of serum-starved medium were seeded in the upper chamber (cell insert), while in the lower chamber 750 μL of basal culture medium (15% FBS). After 24 h of incubation, cells were fixed with 4% PFA, stained with 20 μg/mL of Alexa Fluor 546-conjugated Phalloidin (Invitrogen) overnight, washed with PBS, and observed under a fluorescence microscope (BZ-X700, Keyence, Osaka, Japan). The total number of migrated cells observed at the bottom of the chamber was counted as an average of four different pictures taken per chamber [26 (link)].