Cells were suspended in medium containing Mexisol, and such cells were seeded in Petri dishes lined with an agar (or agarose) gel base.
Operation method
Program 14.5 Cloning Culture in Mexisol
Principle
Cells were suspended in medium containing Mexisol, and such cells were seeded in Petri dishes lined with an agar (or agarose) gel base.
Materials and Instruments
Noble Agar Difco Fetal Bovine Serum 1.6% Mexisol Move 1. Prepare an agar gel base layer according to steps 1-7 in Experiment 14.4. For more product details, please visit Aladdin Scientific website.
Double concentration medium Growth medium Sterile ultrapure water Sterile conical flasks Straws General purpose containers Petri dishes Water baths Triangular flasks Trays Electronic cell counters Bunsen burners
2. dilute Mexizol (1.6 % Mexizol, 4 Pa-S (4000 cps), dissolved in UPW, on ice) with an equal volume of 2× medium (i.e., Ham'SF12, RPMI1640, DMEM, or CMRL1066. Prepare 2× medium with 10× medium by taking one-half of the desired final volume of medium and adding twice as much serum) to 0.8 %, mix thoroughly, and keep on ice. Dilute mexiletine (1.6 % mexiletine, 4 Pa-S (4000 cps), dissolved in UPW, on ice) to 0.8 %, mix well and keep on ice.
3. Trypsin digest the monolayer of adherent cells, collect the suspension cells or bone marrow cells and count them.
4. Prepare the following dilutions of cells up to a maximum concentration of 1 x 105 /ml.
(a) 1×105 /ml
(b) 1×105 /ml was diluted 3 times 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
Mexisol is viscous and is easier to handle with a syringe without a needle.
5. Label each of the four dilutions on 4 small glass vials or test tubes. Pipette 40 μl of each dilution, including the 1 × 105 /ml dilution, into the respective containers. Add 4 ml of 0.8 % Mechisol medium to each container and mix well with a vortex mixer. If the cells are particularly fragile, use a syringe (for dispensing Mechisol. If the cells are particularly fragile, use a syringe (for dispensing Mexisol. Due to its viscous nature, Mexisol can adhere to the inner wall of the pipette, resulting in inaccurate dispensing) to gently aspirate the solution up and down several times, and then use the syringe to aspirate three more 1 ml from each container onto the corresponding three petri dishes. The final concentrations are shown below: 
(a) 1×105 /ml/dish
(b) 330 /ml/dish
(c) 110 /ml/dish
(d) 37 /ml/dish
6. Place the petri dishes in a humidified incubator and incubate the plants until colonies form. Since colonies will form at the interface of agar and Mexisol, add 1 ml of fresh medium/dish or well after 1 week of incubation, and after 2 weeks discard the old solution and replace it with more fresh medium without affecting the colonization.