Microchannel migration imaging

Microchannel Migration Protocol

Jacobelli Lab

Adapted from 4DCell microchannel guidelines

1. After removing the microchannel dish from the packaging add 3mL of PBS to the microchannel dish. Pipette up and down in each well to help wash out the channels. Be careful not introduce any bubbles into the wells or microchannels.
2. Wash the microchannel dish 3x with 3mL PBS. Incubate 5 min at room temp for each wash. When removing liquid be careful not to aspirate directly inside the wells to prevent the microchannels from drying out or introducing bubbles.
3. Wash the microchannel dish 3x with 3mL of warm imaging media. Incubate 5 min at 37C for each wash.
4. After final wash replace with 3mL fresh warm imaging media and incubate for 15min at 37C.
5. Spin down T cells at 400xg and wash 1x with warm imaging media. Resuspend at 10⁷ cells/mL
6. Aspirate off media but leave media remaining inside the wells. Using a pipette remove 10uL from the desired wells for chemokine. Replace this volume with 10uL imaging media with 1ug/mL CXCL10 (stock in -20 is at 200ug/mL, dilute 1:200 prior to use). Use back pipetting technique to minimize bubbles
7. Remove 10uL from the desired wells for cells (at the other end of the microchannel from the chemokine). Replace this volume with 10uL of T cells at 10⁷/mL (1x10⁵ cells).
8. Add 500uL of warm imaging media to the edge of the microchannel dish. Do not cover the microchannels or wells as this will dilute the chemokine.
9. Incubate the dish for 1hr in a humidified 37C incubator.
10. Microchannels are now ready to be imaged on spinning disk. Typically for 4-8hrs with captures every 30sec. For longer runs an additional 500uL of imaging media can be added to the edge of the dish to prevent drying out.

Imaging media: RPMI without Phenol Red + 2% BSA, + 10mM HEPES

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