SOFT AGAR ASSAY FOR COLONY FORMATION--Protocol 1
Note: All volumes are calculated to
cater for four plates per point.
Base Agar:
1. Melt 1% Agar (DNA grade) in microwave, cool to 40C in a waterbath. Warm 2X RPMI
+ 20% FCS to 40C in waterbath. Allow at least 30 minutes for temperature to equilibrate.
2. Mix equal volumes of the two solutions
to give 0.5% Agar + 1X RPMI + 10% FCS.
3. Add 1.5ml/ 35 m Petri dish, allow to set. The plates can be stored at 4C for up to 1 week.
Top
Agar:
1. Melt 0.7% Agar (DNA grade agarose) in microwave, cool to 40C in a waterbath. (It is important not to exceed 40C, otherwise
cells will be killed). Also warm 2X RPMI + 20% FCS to the same temperature.
2. Trypsinise cells and count. It is very important to
have a positive control line (eg. ras transformed).
3. You require 5,000 cells/plate, therefore you need 20,000/tube. Adjust cell count
to 200,000 cells /ml.
4. Add 0.1ml of cell suspension to 10ml yellow capped centrifuge tubes.
5. Label 35mm petri dishes with base agar
appropriately (it is a good idea to remove the plates from 4C about 30 minutes prior to plating to allow them to warm up to room temperature).
6.
For plating add 3ml 2X RPMI + 10% or 20% FCS and 3ml 0.7% Agar to a tube, mix gently and add 1.5ml to each replicate plate (usually
plate out in triplicate). NOTE: Only do one tube at a time so that agar does not set prematurely.
7. Incubate assay at 37C in humidified
incubator for 10 - 14 days.
8. Stain plates with 0.5ml of 0.005% Crystal Violet for >1 hour, count colonies using a dissecting microscope.
Protocol 2. Soft Agar Assay for Colony Formation
Determination of Cell Colony Formation in a High Content Screening ...
These soft agar
colony formation assays have been widely used to determine growth, ... The use of volume as an assay readout has been shown to be
more ...
www.nature.com/app_notes/nmeth/2006/061209/full/an2019.html -
Colony formation assay (Cytokines & Cells Encyclopedia -
COPE)
A soft agar colony formation assay can be used also to measure the sensitivity of human tumors to anticancer drugs (see: HTCA,
human tumor clonogenic cell ...
www.copewithcytokines.de/cope.cgi?key=Colony%20formation%20assay
Soft Agar Assay from Bioprotocol
v,
bioProtocol. > Lab Protocols. My Protocols ... Soft Agar Assay for Colony Formation ... Melt 1% Agar in a microwave and cool to
40ˇăC in a waterbath. ...
www.bioprotocol.com/protocolstools/protocol.jhtml;jsessionid=MCNETTUR310A3R3FQLMSFEWHUWBNQIV0?id=p1123
From Stewart Lab.
http://www.stewartlab.net/Protocols/Soft_agar_1.htm
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