Soft agar colony formation assay
Reagents
0.5% Agaro ()
0.75g agaro dissolved in 50 ml ddH2O,autoclaved,store in room temperature.
0.3% Agaro
0.35g agaro dissolved in 50 ml ddH2O,autoclaved,store in room temperature.
2XDMEM/F12+2XFCS*+2X antibiotics (power+400mlddH2O+100mlFBS+1gNaHCO3+2ml Antibiotics)
0.005%Crystal Violet
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A. Preparation of ba Agar – All steps Must be done sterilely and u cell cultural grade water.
1. Melt 1.5%Agar in microwave and cool to 40◦C in water bath for 0.5-1hour.
2.Using falcon tubes,warm 2X2XDMEM/F12+2XFCS*+2X antibiotics to 40◦C in waterbath. Allow at least 30 minutes for temperature to equilibrate.(Serum may be replaced by agonist, a 1%fcs media or other possible combinations depending on the experimental plans).
3.Mix equal volumes of two solutions to give 0.75%Agar+1XDMEM+1XFCS+ antibiotics.
4.Add 2-3 ml of mixture from step 3 to each well of 6-well plate and t aside for 5 min to allow agar to solidify玉兰花简笔画(The plates can be stored at 4◦C for up to 1 week-let them sit at room temp for 30 min before using)
B. Preparation of top Agar Note: Certain cell types are nsitive to percentage of top agar and customer to determine if 0.3% or 0.4% agar is best suited to cell type in u.方特东方神话
1.Melt 0.3% Agar in a microwave and cool to 40◦研究生推荐信C in water bath for 0.5-1hour(It is impor
tant not to exceed 40梦想手抄报◦C, otherwi the cells will be killed).Warm 2XDMEM/F12+2XFCS*+2X antibiotics(e note for rum in media as described above)to room temperature.
2.Trypsinize adherent cells to relea them and count the number of cells per ml. It is very helpful to have a positive control for colony formation. Take care that the single cell suspension is obtained.
3.This procedure requires 5,000 cells/well for 6-well plate. By using 20,000/tube,there is enough to plate four wells of each 6-well plate. Adjust the Volume so that the cell count=200,000 cells/ml. For 3T3 cells, 10,000 cells/well for 6-well plate.
4.Add 0.1 ml of cell suspension to 10 ml tubes.
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5.Label 6-well plate ba agar dishes appropriately(from Step3).(If they have been stored ,it is good idea to remove the plates from 4◦C about 30 min prior to plating to allow them to warm up to room temperature)
6. To plate, add 4 ml of 2XDMEM/F12+2XFCS*+2X antibiotics and 4ml 0.7% Agar to a tube of cells from Step 4. Mix gently by swirling and 2ml to each of the three or four replicate plates, and t aside for veral minutes at room temp to let the agar to solidify. Only do one tube at a time so that the agar does not t prematurely. Incubate plates at 37 ◦C in humidified incubator. Add 0.5ml media per well the next day.
7.Incubate plates at 37◦C in humidified incubator for 10 to 30 days. Feed cells 1-2 times per week with cell culture media(0.5ml per week for 6-well plate).
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刀锋教育8.Stain plates with 0.5ml of 0.005% Crystal Violet for more than 1 hour.
9.Count colonies using a discting microscope.
