|The methods in this section cover: |
Shields and Sang Medium 3 (SS3) modified for low serum. Supplemented before use with 2% heat inactivated FCS, 2.5% FE2, 12.5IU/100ml insulin and filter-sterilised through 0.22µm filter. Supplemented medium is referred to as CSM.
* Sigma, unless otherwise stated.
The above ingredients are dissolved in 90ml double-distilled water and the pH is brought up to 6.8 with 1%NaOH before the final volume adjustment is made. We make 2 litres at a time.
For routine culture of Cl8+ cells, some labs use ready-made Shields and Sang medium from Sigma (S3652), supplemented with insulin, serum and fly extract as usual.
Sigma I1882. Make up to 12.5 IU/ml stock solution. Put 10mg in universal, add 0.5ml 0.01N HCl to dissolve. The add 19.5ml D = , mixing on vortex mixer. It will go cloudy, leave it to stand and it will clear. Filter-sterilise the solution through a 0.22µm filter. Store at 4 deg. C for up to 1 month.
Remove the medium and cells from the petri dish using a sterile pasteur pipette. Usually the cells will detach and become suspended just by washing the medium up and down. Transfer the medium and cells to a sterile centrifuge tube. If the cells adhere, wash the plate with 1ml D= transferring this to the centrifuge tube, then put on 1ml 0.1% trypsin diluted in 2mM EDTA in D= , and leave at room temperature for 5 minutes. Add a pipetteful of medium from the centrifuge tube back into the dish, wash it all around, then remove it all to the tube. Spin the tubeful at 300g for 5 mins. Remove the supernatant from the pellet and resuspend the pellet in 1ml fresh medium. Prepare two 5ml size bijou bottles with 0.9ml D=, remove a 0.1 ml sample cells from the centrifuge tube and make two serial dilutions to 100-fold. Count using a haemocytometer. The count in one corner (16 squares) gives you the number of cells x 106 in the centrifuge tube. Calculate the quantity of cells to be added to a new 5ml dish: 3÷count gives x ml of original cell suspension to be added, to seed 3 million cells. Add to 5ml fresh medium in a new 5cm petri dish.
Cells usually seeded at about 1.53x105 cells/cm2
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