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.

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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.

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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

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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.

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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