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transfection of insect cells using cationic lipid reagent

By Danielle Ward,2014-11-11 17:18
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transfection of insect cells using cationic lipid reagent

    Transfection of Insect Cells Using Cationic Lipid Reagent (Cellfectin) [-] | [+] Font

Transfecting Insect Cells with Baculovirus DNA

    Use this procedure to transfect Sf9 insect cells in a 6-well format. All amounts and volumes are given on a per well basis.

    51. Plate 9 x 10 Sf9 cells in 2 ml of Sf-900 II SFM containing antibiotics (i.e. penicillin/streptomycin/neomycin; Cat. No. 15640-055) at 0.5X final concentration. Allow cells to attach for at least 1 hour.

    2. For each transfection sample, prepare complexes as follows:

     a. Dilute 1-2 µg of baculovirus DNA in 100 µl of Sf-900 II SFM without antibiotics.

    ?b. Mix Cellfectin before use, then dilute 1.5-9 µl in 100 µl of Sf-900 II SFM without antibiotics.

    ?c. Combine the diluted DNA with diluted Cellfectin (total volume = 200 µl). Mix gently and incubate for 15-45 minutes at room temperature (solution may appear cloudy).

     3. Remove the growth medium from the cells and wash once with Sf-900 II SFM without antibiotics. Remove the wash medium.

    4. Add 0.8 ml of Sf-900 II SFM to the complexes (Step 2c), mix gently and add to the cells. Incubate cells at 27 ?C for 5 hours.

    5. Remove the transfection mixture and replace with 2 ml of Sf-900 II SFM containing antibiotics. Incubate cells at 27 ?C for 48 hours.

    6. Assay for gene activity at 48 hours post-transfection; harvest virus at 72 hours.

Transfecting Adherent Mammalian Cells

    Use the following procedure to transiently or stably transfect mammalian cells. All amounts and volumes are given on a per well basis.

    1. One day before transfection, plate cells in growth medium without antibiotics such that they will be at the recommended confluence at the time of transfection.

Condition Cell no. Growth med. Format Confluence at

    vol. txfn

    5Transient 1-2 x 10 2 ml 6-well 60-80%

    5Stable 1-2 x 10 4 ml 60-mm 30-50%

    2. For each transfection sample, prepare complexes as follows:

    ?a. Dilute 1-2 µg of DNA in 100 µl of Opti-MEM I Reduced Serum Medium (or other medium) without

    serum.

    ? b. Mix Cellfectin before use, then dilute 2-15 µl of

    ??Cellfectin in 100 µl of Opti-MEM I Medium (or other medium) without

    serum.

    ? c. Combine the diluted DNA with diluted Cellfectin (total volume = 200 µl). Mix gently and incubate

    for 10-15 minutes (15-45 minutes for stable transfection) at room temperature (solution may appear cloudy).

    3. Remove the growth medium from the cells and wash once with 2 ml of growth medium without serum. Remove the wash medium.

    ?4. Add 0.8 ml of Opti-MEM I Medium without serum (1.8 ml for stable transfection) to the complexes (Step 2c), mix gently and add to cells.

    5. Incubate cells at 37 ?C in a CO incubator for 5-24 hours. 2

    6. Replace the medium with 2 ml of growth medium containing serum (4 ml for stable transfection). 7. Transient: Test for transgene expression 24-72 hours post-transfection. Stale cell lines: Passage cells at a 1:5 (or higher dilution) into selective medium 48-72 hours post-transfection.

Transfecting Suspension Mammalian Cells (6-well format)

    1. For each transfection sample, form complexes as in Step 2 of the protocol above, with the

    ??following exceptions: (1) Dilute DNA and Cellfectin into 0.5 ml of Opti-MEM I Medium each and (2)

    Incubate complexes for 15-45 minutes at room temperature.

    62. For each transfection sample, centrifuge 2 x 10 cells and aspirate medium.

    3. Resuspend the cell pellet with the DNA-lipid complex solution (Step 1) and transfer to a well of a 6-well plate. Incubate at 37 ?C in a CO incubator for 2

    4-5 hours.

    4. Add 0.5 ml of growth medium containing 30% fetal bovine serum.

     Note: Add PMA and/or PHA, if desired, to enhance promoter activity and gene expression. 5. The following day, add 1-2 ml of complete growth medium. Assay for transgene expression 24-48 hours post-transfection

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