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        Routine methods for growing the cell lines

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        938

        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 Sourcundefined
        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

         

        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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