May 14, 2020
Anchorage-independent growth assay or Soft Agar assay
- 1Laboratorio Cellule Staminali Post Natali e Terapie Cellulari, IRCCS Istituto Gaslini, Genova, Italy
External link: https://doi.org/10.1371/journal.pone.0244069
Protocol Citation: Marzia Ognibene 2020. Anchorage-independent growth assay or Soft Agar assay. protocols.io https://dx.doi.org/10.17504/protocols.io.bgeajtae
Manuscript citation:
Ognibene M, Pezzolo A (2020) Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma. PLoS ONE 15(12): e0244069. doi: 10.1371/journal.pone.0244069
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: May 14, 2020
Last Modified: May 14, 2020
Protocol Integer ID: 37026
Prepare 5% SeaKem low gelling agarose in distilled water and autoclave it.
Warm 10% serum media in 48 °C water bath
Melt the agar in microwave for some minutes. Be sure to mark the level of agar prior to heating so the level can be brought back up with distilled water, to maintain the correct concentration of agar.
Allow the agar to cool at room temperature for 10 minutes, then place it in the 48 °C water bath.
Make a dilution of agar in prewarmed media for a final concentration of 0.6% agar and considering to pour 4 ml in each p60 plastic plates.
Pour the agar in plates and let them sit at room temperature for 40 minutes to solidify, then place in the 37 °C CO2 incubator to allow the plates to adjust to the proper pH.
Prepare a dilution of agar in prewarmed media for a final concentration of 0.3% agar and considering to pour 3 ml in each p60 plastic plates.
Trypsinize cells and resuspend in a minimal volume of media to count them and make the desired cell dilution.
Add 3 ml of 0.3% agar into appropriate number of conical tubes.
Add the right amount of cells to each tube and, working quickly, gently shake the tube in order to suspend the cells evenly through the agar.
Pour the entire content of tube on top of a previously poured 0.6% solidified agar plate.
Swirl plates gently and let them sit at room temperature for 40 minutes to solidify, then place in the 37 °C CO2 incubator.
After 24 hours, feed cells with 1 ml of 10% serum media and check weekly that they are well hydrated.