Tube Formation Assay

Tube Formation Assay

Protocol (12/05/11)

By: Sterling Mikkelson

Day 1.

                Thaw Matrigel™ (BDbioscience cat. no. 356234) on ice, overnight at 4°C.

Day 2.

**Note**  Pipet tips and 48 well plate should be placed in 4°C for a minimum of an hour prior to coating 48 well plate to prevent Matrigel™ from polymerizing while coating well plate.

Coating Plate:

·         Keep the Matrigel™ solution in an ice bath and remove 300ml using a cold pipet tip.  Quickly and carefully, transfer the matrigel to a 48 well.  Be sure to remove any air bubbles before gel polymerizes.

·         Incubate at 37°C for 1 hour.

Cell Plating:

·         Aspirate media from stock flask (confluence approx. 80%).

·         Add 5 ml 0.25% trypsin and incubate at 37°C for 5 min.

·        Neutralize by adding 6 ml RPMI media to flask, triterate by pipetting, then add to 15 mL centrifuge tube.

·         Spin at 1500 rpm for 5 min.

·         Aspirate supernatant and add 2-4 mL media (depending on pellet size)

·         Dilute as necessary for counting cells using either a hemacytometer or automated cell counter

·         Determine cell concentration (Will need minimum of 20,000 cells/well for 2H11 and 100,000 cells/well for HUVEC)

·         Add calculated cell concentration to each Matrigel™ coated well and incubate 16-18 hrs. at 37°C

      Day 3.

Cell Imaging:

·         Image the well plate using an inverted microscope with camera

·         Use a 2x and 4x objective lens to view tube formation

·         Capture image
           
       *Please leave feedback if you have questions or have found this helpful.  Thank you.
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