Soft agar clone formation assay
Soft agar clone formation assay
Soft agar clone formation experiments can be used in (1) basic research on cell differentiation; (2) efficacy testing of clinical tumor therapy, etc.
Operation method
Soft agar clone formation assay
Principle
Soft agar clone formation assay is mainly used for non-adherent growth of cells, some cells (such as normal fibroblasts) can not proliferate in suspension, not applicable to this method. Some malignant tumor cells, not only can proliferate in the adherent state, but also can proliferate in the suspension state, and their ability to form clones in soft agar reflects their degree of malignancy.
Materials and Instruments
Cell samples Move 1. Take the cells in logarithmic growth phase, 0.25% trypsin digestion, centrifugation to collect the cell precipitate. 2. Complete medium resuspension, appropriate dilution and live cell counting, adjust the cell density to 1×103/ml. 3. 0.5 ml of cell suspension (i.e., 500 cells) was taken and added to the medium to make the volume reach 9.4 ml, and incubated at 37℃. 4. preparation of bottom layer agar. 5% agar was placed in a boiling water bath to melt completely, 1ml was removed and transferred to a small sterile beaker, and when cooled to 50℃, 9ml of pre-warmed 37℃ complete culture medium was added quickly, mixed well and immediately 0.8ml was taken and poured into a 24-well plate, and solidified at room temperature. 5. Preparation of upper agar. Add 0.6 ml of 50% agar at 50°C to 9.4 ml of pre-warmed cell suspension in step 3, mix rapidly, take 0.8 ml immediately and pour it into a 24-well plate and set it to solidify at room temperature. Each well that contains 40 cells, generally each experimental group repeated 3 samples. 6. Cultivate for 2--3 weeks. 7. Put the culture plate under the microscope to count the number of clones, each cell colony contains 50 or more than 50 cells for 1 clone, with the formula colony number = n wells in the total number of cell colonies / n, clone formation rate = number of colonies / number of inoculated cells x 100% clone formation rate, and finally photographed. 8. Statistical analysis. The t-test was performed according to the colony number and clone formation rate to analyze the differences between different groups. Caveat 1. Take the cells in logarithmic growth phase, 0.25% trypsin digestion, centrifugation to collect the cell precipitate. 2. Complete medium resuspension, appropriate dilution and live cell counting, adjust the cell density to 1 x103/ml. 3. 0.5 ml of cell suspension (i.e., 500 cells) was taken and added to the medium to bring the volume to 9.4 ml, and incubated at 37 °C. 4. preparation of bottom layer agar. 5% agar was placed in a boiling water bath to melt completely, 1ml was removed and transferred to a small sterile beaker, and when cooled to 50℃, 9ml of pre-warmed 37℃ complete culture medium was added quickly, mixed well and immediately 0.8ml was taken and poured into a 24-well plate, and solidified at room temperature. 5. Preparation of upper agar. Add 0.6 ml of 50% agar at 50°C to 9.4 ml of pre-warmed cell suspension in step 3, mix rapidly, take 0.8 ml immediately and pour it into a 24-well plate and set it to solidify at room temperature. Each well that contains 40 cells, generally each experimental group repeated 3 samples. 6. Cultivate for 2--3 weeks. 7. Put the culture plate under the microscope to count the number of clones, each cell colony contains 50 or more than 50 cells for 1 clone, with the formula colony number = n wells in the total number of cell colonies / n, clone formation rate = number of colonies / number of inoculated cells x 100% clone formation rate, and finally photographed. 8. Statistical analysis. The t-test was performed according to the colony number and clone formation rate to analyze the differences between different groups. Common Problems 1. When performing soft agar clone formation experiments, first of all, cell counting should be accurate and can be repeated and averaged. Secondly, during the operation of the experiment, many steps may cause contamination, this problem must be noted. 2. when mixing the cell suspension and agar, the temperature of the agar must be grasped, when it is too high, it is easy to cause part of the cells to die, and if it is too low, it is easy to make the local caking. 3. Regarding the choice of how many cells to inoculate, one well of 24-well plate, 30--50 cells are enough. Too little, the result is not accurate; too much, the counting is too troublesome. Of course, this also depends on the cell proliferation ability or the degree of malignancy. When the cell proliferation ability is very strong, you can choose to inoculate fewer cells; when the cell proliferation ability is very weak, you can increase the number of cells inoculated. For more product details, please visit Aladdin Scientific website.
Trypsin Fetal bovine serum GIMSA stain PBS Pure methanol
Petri dishes Incubator Microscope
4. Source Practical Experimental Techniques in Molecular Biology.