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Penicillin-Streptomycin Solution

Usually Ships in 48 hrs.

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.

Celebrity Endorsements

Guzmán, Flavio, MD. "Beta Lactams Antibiotics (penicillins and Cephalosporins) Mechanism of Action.” Medical Pharmacology. Pharmacology Corner, 29 Nov. 2008.

Pitout JD, Sanders CC, Sanders WE Jr. Antimicrobial resistance with focus on beta-lactam resistance in gram-negative bacilli. Am J Med 1997; 103:51.

De Britto A.J., Gracelin D.H.S. and Kumar P.B.J.R, 2012, Pteris biaurita L.: A potential antibacterial fern against Xanthomonas and Aeromonas bacteria. Journal of Pharmacy Research 2012,5(1),678-680


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