Medium
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.
| Recipe: |
mg/100ml |
Source* |
| Aspartic acid |
30 |
A 4534 |
| Threonine |
50 |
T 1645 |
| Serine |
35.2 |
S 5511 |
| Asparaginine |
34 |
A 4159 |
| Glutamine |
60 |
G 5763 |
| Phenylalanine |
25.2 |
P 5030 |
| b-alanine |
25.2 |
A 9920 |
| Histidine |
54.8 |
H 9386 |
| Tryptophan |
10 |
T 0271 |
| Arginine |
50 |
A 3784 |
| Cysteine.HCl |
20 |
C 2529 |
| Lysine.HCL |
84.8 |
L 1262 |
| Proline |
40 |
P 4655 |
| Glycine |
50 |
G 6388 |
| a-alanine |
150 |
A 3534 |
| Valine |
40 |
V6504 |
| Methionine |
25.2 |
M 2893 |
| Isoleucine |
25.2 |
I 7383 |
| Leucine |
40 |
L 1512 |
| Tyrosine |
25.2 |
T 1020 |
| Monosodium glutamate |
786 |
G 5889 |
| Glucose |
1000 |
G 7021 |
|
|
|
| MgSO4.7H2O |
400 |
M 1880 |
| CaCl2.2H2O |
932 |
C 7902 |
| KCl |
260 |
P 5405 |
| NaH2PO4.2H2O |
87.6 |
BDH 30132 |
|
|
|
| T.C. Yeastolate |
100 |
Difco 55 77-15-2 |
| Cloline.Cl |
5.2 |
C 7527 |
| Oxaloactetic acid |
25.2 |
O 7753 |
| BIS-TRIS buffer |
104.8 |
B 6391 |
|
|
|
| Penicillin G. Na |
3.2 |
P 3032 |
| Streptomycin sulphate |
10 |
S 9137 |
* 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.
Insulin
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.
Calcium- and Magnesium- free saline
(D minus minus, or D =)
| NaCl |
8 g/l |
| KCl |
0.2 g/l |
| Na2HPO4 |
2 g/l |
| KH2PO4 |
0.4 g/l |
Passaging cell lines
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.
Cell Seeding Numbers
Cells usually seeded at about 1.53x105 cells/cm2
| 5cm dish |
3x106 |
| 6 well plate |
1.6 x 106 per well |
| 24 well plate |
2.7 x 105 per well |
