TRANSFECTIONS
Cell Culture Reagents:
DMEM (high glucose, w/ glutamine, w/o sodium pyruvate)
HFF growth medium (DMEM + 10% FBS + 1x pen/strep)
1x PBS (sterile)
1x Trypsin/EDTA (TE)
Circular glass coverslips (sterile)
DAY 1: 24h before transfection:
Seed HFF cells in 6-well plates so that they become ~50% confluent on transfection day:
For cells grown to 95% confluency in a T-25: Wash cells with PBS 2x, lift with 0.5ml warm TE,
resusupend with 2.0ml warm HFF growth media. This suspension now contains the equivalent of
25cm2 of cells in 2.5ml or 10cm2/ml. The surface area of each well in a 6-well plate is ~10cm2. Each
well will receive the equivalent of 1cm2 or 100ul of cell suspension in 2.5ml total media. (e.g., for 2
HFF transfection wells, mix 200ul cell suspension with 5ml media, dispense 2.5ml/well.)
DAY 2: Transfection I:
1. Warm appropriate amounts of DMEM and HFF media to 37C.
2. Aliquot DMEM in microfuge tubes for DNA: 100ul/transfection (pool when doing multiples).
3. Aliquot DMEM in microfuge tube for LFA: 100ul x N transfections.
4. Aliquot DMEM in 15ml conical: 1.0ml x N transfections.
5. Aliquot HFF growth medium in 15ml conical: 1.0ml x N transfections.
6. Add DNA to tubes from step 2 & Lipofectamine2000 to tube from step 3:
a. e.g., 100-500ng DNA per transfection (start with 300ng)
b. e.g., 2.5ul LFA2K per transfection
7. Mix gently for 5’.
8. Transfer 100ul from LFA tube to each DNA tube.
9. Mix gently for 15’.
10. During incubation, remove media, wash cells 1x with warm PBS.
11. Add 800ul warm DMEM/well from step 4.
12. Return plates to incubator until the 15’ incubation in step 9 is complete.
13. Add 200ul of DNA/LFA complexes to wells dropwise (gently), using micropipet.
14. Incubate for 3h at 37C, 5%CO2.
15. Add 1.0ml HFF growth medium (from step 5) to each well.
16. Incubate for 20h at 37C, 5%CO2.
DAY 3: Reseeding after transfection:
1. Aliquot appropriate amounts of HFF growth media and warm to 37C.
2. Remove medium from wells, gently wash cells 2x with PBS.
3. Add 150u/well warm TE. Incubate as necessary to lift cells.
4. Add 850ul/well warm medium. Mix well.
5. For 24-well plates/coverslips, mix ~50ul of cell suspension with 1ml media and dispense
onto coverslips, 1ml each. (Mix cells with media first, then add to the wells all at once.)
6. For 12-well plates, mix ~100ul of cell suspension with 1.5ml media and dispense into wells.
7. For 6-well plates, mix ~200ul of cell suspension with 2ml media and dispense into wells.
8. Incubate for 24h at 37C, 5%CO2.
Etc.