Team:Washington/Notebook/Flow Cytometry

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(→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 48-51 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 (~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, and resuspend samples in 1mL PBS + BSA
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#: Spin beads at 1000x g for 1 minute
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#: Spin cells at 17,000x g for 1 minute
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# Record cytometry data again with reduced fluorescence background
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{{Template:Team:Washington/Templates/Footer}}

Revision as of 06:55, 20 October 2009

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Flow Cytometry

  1. Set up overnights of parts 48-51 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 (~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, and resuspend samples in 1mL PBS + BSA
    Spin beads at 1000x g for 1 minute
    Spin cells at 17,000x g for 1 minute
  9. Record cytometry data again with reduced fluorescence background


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