Penicillin-Streptomycin Solution (100x)

(Pen-Strep Solution)

Contains 10,000 units of penicillin and 10,000µg streptomycin in a 0.85% saline solution.

Sterile solution.

Penicillin, the beta lactam, breaks peptidoglycan links in non-resistant cell walls and triggers autolysins which kill the cell. Streptomycin, the aminoglycoside, binds to the ribosomes and inhibits protein synthesis.

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Penicillin-Streptomycin Solution Applications

PC-12 Cell Line Protocol

1. Culturing & Maintenance:
• Remove cells from N2 and rapidly thaw in a 37 C waterbath with constant agitation (Should take about 2 minutes)
• Once thawed, transfer the aliquot into a 15 mL conical tube containing 9 mL of Complete Culture Medium
• Spin at 12500 RPM for 5 minutes
• Remove supernatant and resuspend the cell pellet in 15 mL of fresh Complete Culture Medium in a 60 mm
• Feed cells every other day with a 100% change into fresh Complete Culture Medium

2. Carrying / Plating:
• Once cells are about 80% confluent, aspirate off the culture media and add back 10 mL of fresh Complete Medium
• Use a cell scraper to gently lift cells from the plate
• Triturate with a glass Pasteur Pipette to break up any chunks and plate into new 60 mm dishes to carry, or into experimental dishes
• To carry, cells should be diluted to anywhere between 1x10 4 cells/mL and 3x10 4 cells/mL Normally the cell count (via hemo cytometer) after scraping / triturating is around 18 which is 18x10 4 cells/mL
• (18x10 4 cells/mL)(x) = (1.5x10 4 cells/mL)(15mL)
• For experiments, the density you plate at depends on the assay, etc...

3. Differentiating:
• Remove Complete Culture Medium and replace with differentiating medium. DMEM containing 1% horse serum, 1% Pen-Strep and 100ng / mL NGF
• Feed cells every other day with differentiating medium until ready for experiment

4. Medias:
Complete Culture Medium (500 mL):
• 445 mL of DMEM
• 25 mL of horse serum
• 25 mL of calf serum, 5 mL Pen-Strep
Differentiating Medium (50 mL):
• 48.5 mL of DMEM
• 500 uL NGF
• 500 uL Horse Serum
• 500 uL of Pen-Strep

Transfection using Lipofectamine

1. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Generally need to have 2 μg of DNA for each tranfection. Purify the linear fragment using any appropriate method.

2. Day 1) Plate cells in 6 well plates in 2 ml media (can have pen/strep). Plate an amount that will result in approximately 85 to 90% confluence (see table below) on the day of transfection.

3. Day 2) Prepare transfection cocktail. In Tube "A" place 2 μg of DNA in Opti-Mem-I for a final volume of 200 μl.

4. In tube "B" place 190 μl of Opti-Mem-I and 10 μl Lipofectamine 2000. Incubate for 5 minutes at room temperature, but not more than 10 minutes

5. Gently combine tubes A and B and incubate at least 20 minutes, but can be up to several hours.

6. Remove media from 6-well plates and add 1.6 ml of fresh media (which can contain pen/strep).

7. Add the lipofectamine/DNA mixture to each well and gently swirl to mix.

8. Incubate at 37°C for 4 to 6 hours for delicate cells or overnight for tough cells.

9. After incubation, remove media and replace with fresh media. If the transfected DNA contains a fluorescent protein, check for transfection efficiency by viewing in a fluorescent microscope. Allow cells to grow for 1 to 2 days.

10. Trypsinize cells and transfer to a 10 cm plate and add selecting antibiotic

11. Incubate in tissue culture incubator for 4-6 hours or overnight.