Fluospheres amine modified microspheres

To prepare the Agarose gel-matrix, 1 g of agarose was dissolved in 100 ml of distilled water to prepare $1\%$ gel. The mixture was boiled at $121{}^{\circ }$ C and subsequently filtered using syringe filter (Biofil) while it is still hot. The solution is then allowed to cool at $50{}^{\circ }$ C. $20\mathrm{\mu }$ L of both the beads are mixed and resuspended in 1 mL of liquified agarose solution. The beads were purchased from Invitrogen with specific excitation-emission characteristics. They are, Invitrogen FluoSpheres Amine-Modified Microspheres, $1.0\mathrm{\mu }$ m, yellow-green fluorescent (505/515) ( Catalog number: F8765), and Invitrogen FluoSpheres Carboxylate-Modified Microspheres, $1.0\mathrm{\mu }$ m, Nile Red fluorescent (535/575), (Catalog number: F8819). The filtered liquid mixure is then poured in a capillary tube (Z114995, SigmaAldrich) of volume $100--200\mathrm{\mu }$ L) and left to dry for 10 min at room temperature.

Red carboxyl-functionalized particles 20 nm and 200 nm (FluoSpheres® carboxylate modified microspheres, Invitrogen), 200 nm red amine-functionalized particles (FluoSpheres® amine-modified microspheres, Invitrogen) and non-functionalized 200 nm red-dyed polystyrene particles (Fluoro-Max R200, Thermo Fisher Scientific) were used. The particles were characterized in DMEM w/o FBS and in SLF. SLF was prepared by adding 385 μL stock solution of DPPC (13 mg/mL in ethanol) to 25 mL of a preheated (37 °C) DPBS with calcium and magnesium (Thermo Fisher Scientific).