Flow Cytometry

96-well plate spinning

Note

It is convenient to prep cells for flow cytometry in a 96-well plate because you can add media and do mixing with a multichannel pipette.

Important

Make sure you have at least 2 autoclaved tips per well (1 for triturating, 1 for transferring to U-bottom). For a full 96-well, that means at least 2x200 µL boxes. Plus more for PBS washes and adding in dissociation media!

  1. Wash, dissociate, and triturate cells in plate (swap out tips every time for the trituration).

    • You don’t need to swap tips if you hover above the plate while adding in wash and dissociation buffer but you must swap tips for trituration (i.e. everytime you put the tip into the well)

  2. Take the plate out of the BSC (the plate is no longer sterile at this point).

  3. Optional Transfer contents of (flat bottom) plate to a 96-well U-bottom or V-bottom plate

    • A U-bottom plate is not necessary, but cells pellet better on U- or V-bottom plates, and lose less volume running flow.

  4. Spin the plate at 400 rcf (g) for 5 min using the plate bucket rotor.

    • Balance with another 96-well plate filled with roughly equivalent amount of water (there’s a balance plate near the centrifuge) but balancing doesn’t have to be exact at this low speed

  5. Aspirate supernatant.

    • Tilting the plate and aspirating from corner helps to avoid disturbing the cell pellet. Do NOT aspirate straight from the middle of well.

  6. If performing any staining (e.g. Live/Dead, surface markers, intracellular, etc.), skip step 7 and proceed to Cell staining for flow.

  7. Resuspend in ~200 µL PBS and pipet up and down to mix well (must be single-cell suspension—no clumps!).

    • Swap tips in between each transfer to avoid contamination between wells!

    • If using flat-bottoms, decrease the Attune uptake volume settings so you don’t create bubbles (i.e. you will lose more volume with flat-bottom wells)

  8. Load plate into the Attune CytKick autosampler and run.