Smolke:Protocols/Preventing Clogs

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Quanta clogging can be acute or chronic. Sudden and severe clogs are obvious and the source easy to identify, whereas culprits for the slow build-up of obstructions are much harder pinpoint. Therefore, everyone should participate in the effort to prevent Quanta clogs, regardless of the cell type used. The Basic Measures listed below are simple steps that everyone should do. Certain cell types (e.g., HEKs) are known to be sticky and frequently experience severe clogs. Users who run such samples should take extra steps (see suggestions below) to prevent clogs. As people try out these different tips, please report back (either here on the Wiki or to Yvonne/Kathy on what seems to work and what doesn't).

  • Basic Measures (i.e., everyone should do this, regardless of cell type)
    • Perform start-up and shutdown procedures as described by the start-up/shutdown wizard
    • Analyze cells using V- or round-bottom plates
    • Set re-dispense setting at "High" intensity for 12 cycles
    • Make sure the event rate ("Rate" shown at the upper left-hand corner on the screen) is below 1000; dilute your samples if necessary
  • Additional Suggestions
    • Keep cell density below 1 million/ml.
    • Avoid over-trypsinization (i.e., letting cells sit in trypsin for too long or not neutralizing with sufficient volumes of media). Over-trypsinization releases free DNA into the prep, which will string everything together.
    • Set up your worklist such that Coulter Clenz is routinely run through the system as a preventive measure. To do so, you could either have Coulter Clenz in your sample plate or leave it in a cup. When setting up the worklist, insert the Coulter Clenz well/cup throughout your sample list (e.g., every 12th sample).
    • Under "Current Settings," check the "Long Rinse" box (top right area of screen). This will force a longer rinse between each sample.
    • Under "Current Settings," change the "Throughput" setting (also in top right area of screen) from High to either Medium or Low. This will make the system flush the flow cell more frequently (once after every 8 samples for High, every 3 samples for Medium, and every sample for Low).
    • Filter the cells through a 60-70-um mesh or cell strainer to remove larger lumps.
    • Keep cells suspended in 1-5 % BSA/media helps prevent clumping.
    • Try adding some EDTA (0.02%).
    • Try adding DNase at 50 U/ml in all preparation media.
    • Accutase sold by Phoenix Flow Systems (San Diego, CA) prevents clumping. They also sell Accumax, an enzyme-based product used for creating single cell suspensions from tissue samples. It is used to disaggregate clumpy cells and viability seems to be very good after treatment.