Facsaria special order research product flow cytometer sorter

The procedure for microsphere preparation was modified from an existing protocol [39 (link)]. Protein A coated and polystyrene microspheres measuring 10 μm in diameter, were purchased from Bangs Laboratories (Fishers, IN). L-selectin or hIgG proteins were saturated on the surfaces of the microspheres by first incubating the microspheres in 1% BSA / DPBS+ for 30 minutes at room temperature; subsequently microspheres were incubated in tris balanced saline (TBS, pH 8.2) supplemented with L-selectin or hIgG for 30 minutes at room temperature. Following the incubation in L-selectin or hIgG, microspheres were blocked in 1% BSA / DPBS+ for one hour at room temperature and stored at 4^°C.
Microspheres were characterized using flow cytometry as previously described [40 (link)] to determine the incubation concentration at which the microspheres were saturated with L-selectin, such that the microspheres presented the maximum level of L-selectin allowed by their binding capacity (S1 Fig). L-selectin and hIgG microspheres were washed with 0.1% BSA / DBPS+ and incubated with fluorophore conjugated antibodies for 30 minutes at room temperature using a one-step protocol. Subsequently, L-selectin and hIgG microspheres were washed in DBPS+ and analyzed using a FACSAria Special Order Research Product flow cytometer/sorter (BD Biosciences).

Polystyrene microspheres from Bangs Laboratories (Fishers, IN) with a mean diameter of 10 μm were conjugated using a technique previously described^{51 (link)}. In brief, microspheres were washed in TBS pH = 4.0, twice in DPBS, and then incubated at 2.5 × 10⁷ microspheres/mL in the desired molecular probe in DPBS at equimolar concentrations (relative to 10 ug/mL of recombinant human P-selectin Fc chimera). Conjugated microspheres were washed then blocked with 1% BSA,1% FBS in DPBS. Immediately prior to conducting DBTA, microsphere conjugation was verified via flow cytometry where 100,000 microsphere samples were incubated with the appropriate fluorophore-conjugated antibody or control (e.g., PE-conjugated mouse anti-human CD62P or PE-conjugated mouse IgG1) at 5 µg/mL concentration in 0.1% BSA, DPBS for 30 min. These microspheres were washed twice with 1% BSA and once in DPBS then resuspended in DPBS and analyzed by a FACSAria Special Order Research Product flow cytometer/sorter (BD Biosciences, San Joes, CA). Prior to perfusion in the dynamic biochemical tissue analysis, DBTA probes (conjugated microspheres) were resuspended, unless noted otherwise, at a concentration of 5 × 10⁵ probes/mL in DPBS and verified with a Scepter 2.0 handheld, automated cell counter (EMD Millipore).

Using a FACSAria special order research product flow cytometer/sorter (BD Biosciences, San Jose, CA), surface molecular expression levels of CD44 (2C5), sLe^A (116-NS-19-9), sLe^X (CSLEX-1), and HECA-452 antigen were analyzed on the nontreated and treated lung cancer cell lines. Cell suspensions at a density of 10⁷ cells/mL were incubated with 10 μg/mL of antibodies or with their matched isotype control antibodies for 30–45 min at 4°C. Cells were washed three times with 0.1% bovine serum albumin (BSA) in DPBS⁺ and were incubated with secondary antibodies for 30 min at 4°C. Cells were washed again twice with 0.1% BSA/DPBS⁺, followed with one DPBS⁺ wash, and then were resuspended in DPBS⁺ before running through the flow cytometer.