Flow Cytometer, BD Accuri

BD ACCURI FLOW CYTOMETER (B457)

Rules and Guidelines

Before use:

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 tuan_m_nguyen@bd.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.

Fluidics:

• 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

Optics:

• 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.)

Start-up:

• 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.

Analysis:

• 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.

Clean up:

• 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

Quality Control

• 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

in.

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)