Clonal culture in agar
Summary
The agar is liquid at high temperatures, but becomes a gel at 37°C. Cells are suspended in warm agar gel medium and cultured after the agar has formed a gel, forming scattered colonies that can be easily separated.
Operation method
Scheme 14.4 Cloning culture in agar
Principle
The agar is liquid at high temperatures, but becomes a gel at 37°C. Cells are suspended in warm agar gel medium and cultured after the agar has formed a gel, forming scattered colonies that can be easily separated.
Materials and Instruments
Noble Agar Difco Fetal Bovine Serum
Double concentration medium Growth medium Sterile ultrapure water Sterile conical flasks Straws Universal containers Petri dishes Water baths Triangular flasks Trays Electronic cell counters Bunsen burners
Move
1. Number or mark the side of the petri dish base and place the petri dish conveniently in the tray.
2. Prepare 2× medium with 40 % FBS (i.e., Ham'SF12, RPMI1640, DMEM, or CMRL1066. Prepare 2× medium with 10× medium by taking half the amount of the desired final volume and adding twice the concentration of serum) and keep it at 37℃.
3. Weigh 1.2 g of agar.
4. Add 100 ml of sterile UPW to a sterile conical flask and another sterile flask. 1.2 g of agar should be placed in the conical flask, capped and boiled for 2 min. Alternatively, autoclave the agar in advance. However, if it has been stored, it should still be dissolved by boiling or melted in the microwave for use.
5. Place the boiled agar and the UPW bottle in a 55°C water bath.
6. Prepare a 0.6 % agar base layer by mixing equal parts of 2 x medium and 1.2 % agar and keep at 37°C. If any growth factors, agar, or other ingredients are required, they should be added to the base layer. If any growth factors, hormones or other additives are required, they should be added to the base layer of agar medium (Growth Medium, 1×, for cell dilution) at this time. 
7. Add 1 ml of 0.6 % agar medium to the Petri dish and shake well to ensure that the medium covers the entire bottom of the dish. Place at room temperature to set. 
8. Prepare cell suspension and count.
9. Prepare 0.3% agar medium and keep at 37°C. This medium can be used at 37°C. This medium can be prepared by mixing 2× medium at 37°C, 1.2 % agar at 55°C and UPW at 55°C in a 2:1:1 ratio.
10. Prepare the following dilutions of cells to a maximum concentration of 1 x 105 /ml.
(a) 1×105 /ml
(b) 3-fold dilution of 1×105 /ml to 3. 3×104 /ml
(c) Dilute 3. 3×104 /ml 3 times to 1. 1×104 /ml
(d) Dilute 1. 1×104 /ml 3 times to 3. 7×103 /ml
11. Label the concentrations of the four dilutions on four small glass vials or test tubes. Pipette 40 μl of each dilution, including 1×105 /ml of cell solution, into the corresponding containers at 37°C, add 4 ml of 0.3% agar medium, mix well, and then pipette three 1 ml of each container (general-purpose containers, small glass vials or centrifugal tubes, for diluting the cells) and add them to the corresponding three petri dishes. Petri dishes. The final concentrations are shown below:
(a) 1×103 /ml/dish
(b) 330 /ml/dish
(c) 110 /ml/dish
(d)37 /ml/dish
Ensure that the top layer of agar medium has sufficient time to cool to 37°C before adding cells.
12. Allow the solution in the Petri dish to form a gel at room temperature.
13. Place the dish in a clean plastic box with a lid and incubate for 10 days at 37°C in a humidified oven.
Caveat
Before preparing the medium and cells, the cell dilutions were calculated and labeled on the dishes, and three dishes were prepared for each dilution of cells when testing the clone formation rate of a cell line. For each 3.5 cm dish, the appropriate number of cells to be inoculated is 1000, 333, 111, and 37 cells. This is a 3-fold dilution of the cell suspension in turn. If growth factors, hormones or other additives are to be added, they should be added to the lower layer of 0.6 % agar.
Common Problems
If it is necessary to determine the titer of growth factor activity or to select a different factor, add the desired amount of factor to the petri dish before spreading the bottom agar.
For more product details, please visit Aladdin Scientific website.