Team:Washington/Notebook/Flow Cytometry

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==== Flow Cytometry ====
==== Flow Cytometry ====
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# Set up overnights of parts 48-51. Let grow overnight.
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# Set up overnights of parts J36848-J36851 in terrific broth (TB). Let grow overnight at 37°C.
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# Dilute 1 ul overnight into 1ml
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# Dilute 20 μL overnight into 1 mL TB and grow at 37°C until OD 0.3 to OD 0.8.
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# Add 1 mm IPTG and let grow for four hours
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# Add IPTG to 1 mM concentration and let grow for minimum of four hours at room temperature (~23°C).
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# After cells have grown up, place in flourophore (1 um) and allow time to bind (1 hour)
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# 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).
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# Also place 1 ul beads in 1 ml along with 1 ul flourophore.
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# Preincubate second set of samples in 1 μM biotin to test for nonspecific binding of the fluorophore to the cells
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# Allow beads to bind to flourophore, then spin beads down, remove supernatent and replace with 1 ml water.
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# Similarly incubate streptavidin-coated beads with fluorophore in PBS + BSA.
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# Read the samples through the flow cytometer
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# Record cytometry data with 100 μL of each sample.
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# Centrifuge samples, carefully pipette off supernatant to ~20 μL to avoid disturbing loose pellets, and resuspend samples in 1mL PBS + BSA
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#*: Spin beads at 1000 x g for 1 minute
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#*: Spin cells at 17,000 x g for 1 minute
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# Record cytometry data again with reduced fluorescence background
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==== Flow Protocol References ====
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#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]
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#Cell division in ''Escherichia coli'' cultures monitored at single cell resolution. Roostalu J et al. [http://www.biomedcentral.com/1471-2180/8/68 BMC Microbiology 2008, 8:68; doi:10.1186/1471-2180-8-68]
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Latest revision as of 07:13, 20 October 2009

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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 References

  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]
  2. Cell division in Escherichia coli cultures monitored at single cell resolution. Roostalu J et al. [http://www.biomedcentral.com/1471-2180/8/68 BMC Microbiology 2008, 8:68; doi:10.1186/1471-2180-8-68]
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