Dynamin cells

 

Maintenance Medium:

            DMEM (high glucose, +sodium pyruvate, Gibco # 41966-029)

            10% FBS

            1% PenStrep

            400 µg/ml G418 (Geneticin, Gibco, now SIGMA,

                                               50 mg/ml stock, top of the fridge

                                               dilute 1:125, use sterile 1ml plastic pipette)

            1 µg/ml tetracycline (Sigma)-> stock:2 mg/ml in freezer,

                                                           dilute 1:2000, use sterile filtertips

            200 ng/ml puromycin (Sigma, P7255 or P8833) -> stock: 200 µg/ml

                                               dilute 1:1000, use sterile filter tips

 

I use our normal HeLa medium (i.e. DMEM, 10% FBS, 1% Pen/Strep, 1% MEM) and supplement the amount  needed with G418, Puromycin and Tetracycline just before use. Thaw a new aliquot of Puromycin and Tetracycline every two splits.

 

To split the cells, proceed as with Hela's. Do not let the cells become too confluent. The cell bodies should not touch each other at the time of splitting. In this case, the cells will easily disperse after trypsinisation just by vigorous tapping against the side of the flask. If cells are clumpy, titriate by pipetting up and down with a 10 ml pipette. Split the cells 1:5 every two days.

 

Freezing medium:

            DMEM

            5% DMSO

            15% FBS

            10 mM Hepes pH 7.4

            3 vials per 10 cm dish

 

Induction:

            Wash cells 3-5x with large volumes of PBS before trypsinising.

            Split cells and replate in medium without tetracycline.

            Add tetracycline to those dishes that require medium.

            Split 1.5 x 10e6 cells per 10 cm dish.

            Set up one 6well dish with coverslips to monitor induction

            by immunofluorescence, use 1.5x10e6 cells/6well

            Exchange medium the next morning, to remove all traces of             tetracycline from induced cells

           

            Grow cells a total of 48 hours in the absence of tetracycline

            Cells ought to be no more than 75% confluent on the day of the             experiment; cells should not touch each other and there should be plenty of space between the cells!!!

           

            To check induction, incubate cells on one coverslip (-/+ Tet each)

            for 15 min with biotinylated Transferrin at 25 µg/ml in DMEM.

            Fix all cells on coverslips with PFA as usual, permeabilise with 0.2%             Triton- X100 in PBS and stain either with HA/EEA1 followed by             TexasRed/OregonGreen or with HA alone (for those coverslips that             were exposed to Transferrin) followed by Streptavidin-OregonGreen.

            Determine % of cells expressing HA-K44A and % of cells taking             Transferrin up into the perinuclear recycling compartment.