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Cl8+
Growing
the Cells

The methods in this section cover:



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.

Methods in this section


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.

Methods in this section


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

Methods in this section


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.

Methods in this section


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

Methods in this section



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If you have any comments or queries, please e-mail mjm5@st-and.ac.uk or dc6@st-and.ac.uk