Transfection of COS1 Cells by DAVID MANN Method:

Transfection of COS1 Cells by DAVID MANN Method:

Dominik Haudenschild and Mehrdad Tondravi, 1991 at CBRC

Day 0: For 6 well plates seed cells @ !x105.

Day1: i) Remove medium.

ii) Wash cells with 1X TBS containing 1.0 mM Ca++ and 0.5 mM Mg++.

iii) Incubate each well in 1000 µl of TBS+Mg+Ca+DNA+DEAE-Dextran.

PREPARE DNA-DEXTRAN IMMEDIATELY PRIOR TO USE:

ADD IN ORDER DNA to 1 ml of TBS, then add 10 µl of 100X DEAE-Dextran

100x DEAE-Dextran= 50mg/ml

iv) Incubate at 37° C for 30 min. (IF CELLS START TO ROUND UP REMOVE DNA).

v) Remove DNA, Add 1 ml of serum-free DMEM to wash away DEAE-Dextran.

vi) Incubate cells in 1 ml of DMEM+ 10% FBS+ 100 µM Chloroquine @37° C for 3Hrs.

During this time check the cells to make sure that they are not rounding and coming off of the plate. IF THIS HAPPENS, REMOVE THE MEDIUM AND WASH THE CELLS.

vii) Remove and discard medium.

viii) Wash cells 2X with TBS+Ca+Mg or with serum-free DMEM.

ix) Incubate cells with 2 ml of DMEM+ 10% FCS. Maximal expression of the transfected gene is supposed to be between 48 and 72 hrs. post transfection.

x) FOR LABELLING CELLS: After 24-36 Hrs. replace medium with labeeling medium.

a) METHIONINE LABELLING: Use Met-free DMEM+ 10% FCS and upto 50µCi/ml of Met.

b) GAG LABELLING: Use SUlfate-free DMEM + 5% DIALYSED FCS (dialyzed against PBS) + Glu + Pyruvate + 1.0 mM Ca + 0.5 mM Mg + non-essential AAs + 100µCi/ml of SO4. DO NOT ADD PEN/STREP BECAUSE THEY ARE A HIGH SOURCE OF SULFATE!!

Label for as long as 36 hrs.

Mark Williams' version:

1) Wash cells 2X with TBS+Ca+Mg

2) Incubate with DNA for 45 Min. @37° C.

3) Wash DNA from cells 2X with TBS.

4) OPTIONAL: Shock cells with HBS+ 10% DMSO for 2 Min. Then Wash Cell 3X

5) OPTIONAL: Shock with DMEM+ 5% FCS + 100µM Chloroquine @ 37° C for 3 Hrs.

LOPATA, CLEVELAND AND SOLLNER-WEBB VERSION:

Day 0) Seed cells @ 6x105 cells/60 mm dish. DMEM + 10% FCS

Day 1) Wash Cells with 2.5 ml DMEM without serum

Replace with 1.5 DMEM + Supercoiled DNA + DEAD-Dextran. FOUND THAT 8-16 µg/ml of DNA AND 200 µg/ml of DEAE-Dextran works best.

Incubate for 4 hrs. @ 37° C in 5% CO2 incubator.

Remove DNA sol'n and replace with HBS+ 10% DMSO. INCUBATE @RT FOR 2 MIN., but upto 30 min. is still OK.

Wash cells 2X with PBS and incubate in 5 ml of DMEM +10% FCS for up to 72 hrs.

Solutions:

10X TBS:

NaCl 80 g

KCl 3 g

Na2HPO4 1 g

Dextrose 10 g

Tris-Base 30

Bring to 1 l with H2O after adjusting to pH 7.4 with HCl.

10X HSB:

NaCl 80.06 g

KCl 3.78 g

Na2HPO4 1.88 g

Dextrose 10.81 g

Hepes 50.04 g

Bring to 1 L after adjusting the pH to 7.1