Team:Washington/Notebook/Flow Cytometry

Flow Cytometry

1. Set up overnights of parts J36848-J36851 in terrific broth (TB). Let grow overnight at 37°C.
2. Dilute 20 μL overnight into 1 mL TB and grow at 37°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°C).
4. Record OD 600 and add equivalent of 1 μL 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 μM 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 μL of each sample.
8. Centrifuge samples, carefully pipette off supernatant to ~20 μL 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

9. Record cytometry data again with reduced fluorescence background

Flow Protocol Reference

1. Isolating and engineering human antibodies using yeast surface display. Chao G et al. [http://www.ncbi.nlm.nih.gov/pubmed/17406305 Nat Protoc. 2006;1(2):755-68]