Team:Washington/Notebook/Flow Cytometry

Flow Cytometry

 * 1) Set up overnights of parts J36848-J36851 in terrific broth (TB). Let grow overnight at 37&deg;C.
 * 2) Dilute 20 &mu;L overnight into 1 mL TB and grow at 37&deg;C until OD 0.3 to OD 0.8.
 * 3) Add IPTG to 1 mM concentration and let grow for minimum of four hours at room temperature (~23&deg;C).
 * 4) Record OD 600 and add equivalent of 1 &mu;L of OD 2.0 cells to 1mL PBS with 1 mg/mL BSA and specified flourophore concentration (0-100 nM) and allow time to bind (30 minutes to 1 hour).
 * 5) Preincubate second set of samples in 1 &mu;M biotin to test for nonspecific binding of the fluorophore to the cells
 * 6) Similarly incubate streptavidin-coated beads with fluorophore in PBS + BSA.
 * 7) Record cytometry data with 100 &mu;L of each sample.
 * 8) Centrifuge samples, carefully pipette off supernatant to ~20 &mu;L to avoid disturbing loose pellets, and resuspend samples in 1mL PBS + BSA
 * Spin beads at 1000 x g for 1 minute
 * Spin cells at 17,000 x g for 1 minute
 * 1) Record cytometry data again with reduced fluorescence background

Flow Protocol References

 * 1) Isolating and engineering human antibodies using yeast surface display. Chao G et al. Nat Protoc. 2006;1(2):755-68
 * 2) Cell division in Escherichia coli cultures monitored at single cell resolution. Roostalu J et al. BMC Microbiology 2008, 8:68; doi:10.1186/1471-2180-8-68