Jun 28, 2022
Colony formation assay in Matrigel
- 1The Francis Crick Institute
Protocol Citation: Goran Tomic 2022. Colony formation assay in Matrigel. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g747k3gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 28, 2022
Last Modified: June 28, 2022
Protocol Integer ID: 65555
Keywords: Matrigel, colony formation
Abstract
This is a colony formation assay that I have set up as an alternative to soft-agar assay. It is based on a 3D on-top assay (Lee et al. 2007, 4, 4, 359) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933182/
Attachments
Thaw Matrigel on ice at 4 C overnight. Prechill one 48-well plate, 200 uL box of tips.
Coat prechilled culture surface with a thin layer of Matrigel (80 uL per well in a 48-well plate)
Incubate for 20 min at 37C to allow to gel
Trypsinise cells (Filter through a 40 um mesh to make single cells)
Aliquot cells in a 1.5 mL eppendorf tube to have 2500 cells/100 uL, multiplied by the number of replicates, and spin down at 1200 rpm. Depending on the cell line, optimisation of cell number might be needed.
Add 100 uL of cell suspension on top of Matrigel in wells.
Leave for 20 min at 37C for cells to attach to Matrigel.
Chill the remaining medium on ice and add Matrigel to 10%
Gently add 100 uL of the 10% Matrigel medium on top
Maintain culture for 4 days. Change the medium every 2 days (with 10% Matrigel)
Observe the formation of colonies. Once ready for imaging, scan on Oxford Optronics colony counter or another method of choice. https://www.oxford-optronix.com/gelcount-cell-colony-counter
It helps to top up the wells with PBS or medium to reduce the liquid meniscus interfering with the scanning.