Flow Cytometer, BD Accuri
BD ACCURI FLOW CYTOMETER (B457)
Rules and Guidelines
1. New user MUST be trained by the captain or present users. For specific questions, you can ask Katie in B457 or email Andrew (BD representative) at email@example.com.
Flow Cytometer User Instructions
Notes from the Training Session:
• Do not run cells > 40um in diameter (the size of the flow cell is 200um and multiple cells sticking together can lead to clogging)
• The set threshold is sufficient for cells at least the size of lymphocytes. If you are running bacteria, you will need to change this.
• ONLY USE BD APPROVED LIQUIDS (they are found under the hood of the accuri)
• Sheath fluid = 1L of di water + 1 vial of bacterial static reagent
• Cleaning concentration (detergent) = 3mL concentrate + 197mL di water
• Decontamination solution (special bleach) = 20mLs concentrate + 180mLs di water
• Fluidics works best with 1 million particles/500uLs. If you cells are especially large or sticky, use less.
• "Slow" fluidics: recommended for DNA stains, forms a uniform stream in the flow chamber
• "Fast" fluidics: stream is larger leading to less resolution
• Blue laser is ON, yellow/green laser is modulated. This helps the machine know which laser is which.
• Alternative filters can be purchased for special applications
• Voltage may not be change. (Voltage is fixed to optimize sensitivity.)
• Make sure there is sufficient sheath fluid and enough space in the waste container
• Right after turning machine on, run di water for 15 mins (FAST fluidics). This purges all the air bubbles to insure sensitivity.
• If you are suspicious of the cleanliness, can also go to Instrument > Run Clean Cycle. This will run cleaning solution through the fluidics and is usually not necessary if users have been cleaning the
machine after every use.
• Use the analyze and statistic tabs to make blots and analyze data
• To export plots, drag and drop onto the desktop (to get higher resolution, change drag and drop options)
• To export raw data, File > Export All Samples To Third Party. This exports the data as an FCS file.
Compensation: required if you are looking at fluorphores with overlapping fluorescence
• Need to compensate manually: run a single color control, separate out the "single +" and "double +" cells using the quadrant marker, show median in the statistics bar, click set color compensation, then
correct by subtracting a few percentage points so that the "double +" are in line with the single positives. Apply to all samples.
• Run decontamination solution for 2mins.
• Then run di water for 2mins.
• Click Instrument > Run cleaning fluid cycle
• Press off–> the instrument then goes through its own decontamination process for 20mins.
If clogged: you will know that it is clogged because you will run a sample and get no events. Or you will run a sample and get a very small event rate.
• Use Backflush to gently unclog machine
• Use Unclog for a more powerful unclog
• Use 8-peak validation beads (found in 4C cold room): 2 mins, slow flow rate, gate on the singlet beads
• Check histogram of 4 different filters. You expect to see 8 peaks on FL1, FL2 and FL4. FL3 does not have 8 peaks usually.
• The CV (coefficient of variation) should be lower than 6% indicating that the peak is tight.
Maintenance (every 2 months for 2 moth maintanence kit or 1 year maintenance kit)
• Filters: change filters for the sheath fluid (large filter), decon and cleaning solution (smaller filters)
• Sheath in line filter: Look under container storage for this filter.
• Peristallic pump (important for accurately measuring volume): push out grey tab to pull out cover, unscrew blue connections, take out tubing, replace with new by realigning the track and snapping new pump
MANUFACTURER: BD Biosciences
WARRANTY: One year of warranty comes with the C6. Since this is a special order C6 WE have on site support from a field service engineer.
CONTACT: If you have any instrument issues please call tel:877.232.8995 prompt 2 for a service engineer.
FULL MANUAL: File:BD Accuri C6Flow Cyto Instrument Manual.pdf
--Jessica Metzger 14:42, 21 April 2015 (EDT)