Preparation of 10X Dulbecco’s Phosphate buffered saline (DPBS)

Overview:

• Phosphate buffered saline (PBS) is a buffer solution commonly used in biological research.
• PBS supports cell viability by keeping the medium pH and osmolality near-physiological.
• Several formulations of PBS have been described. Most formulations contain similar amounts of sodium chloride and potassium chloride but differ in the concentration of Na2HPO4 and KH2PO4. PBS can also be supplemented with Calcium chloride and Magnesium chloride. Some of the PBS formulations are_ _ _ _ _
  • Sambrook’s PBS (1X) : 137 mM Sodium chloride, 2.7 mM Potassium chloride, 10 mM Disodium hydrogen phosphate, 1.8 mM Potassium dihydrogen phosphate
  • Dulbecco’s PBS (1X) : 137 mM Sodium chloride, 2.7 mM Potassium chloride, 8.1 mM Disodium hydrogen phosphate, 1.47 mM Potassium dihydrogen phosphate
• Here we describe the procedure to prepare 10X Dulbecco’s PBS. Working solution can be prepared by diluting the stock solution 10 times i.e., to prepare a 100 ml working solution, take 10 ml stock solution and mix with 90 ml water.

REQUIREMENTS

Reagents and solutions
Sodium chloride (NaCl)
Potassium chloride (KCl)
Disodium hydrogen phosphate, anhydrous (Na2HPO4)
Potassium dihydrogen phosphate (KH2PO4)
Deionized / Milli-Q water

Equipment and disposables
Measuring cylinder
Conical flask / Beaker (2L capacity)
pH meter and/or pH paper
Magnetic stirrer with heat control (optional)

Composition of 10X Phosphate buffered saline (PBS)
1370 mM Sodium chloride
27 mM Potassium chloride
81 mM Disodium hydrogen phosphate
14.7 mM Potassium dihydrogen phosphate

Composition of 1X PBS
137 mM Sodium chloride
2.7 mM Potassium chloride
8.1 mM Disodium hydrogen phosphate
1.47 mM Potassium dihydrogen phosphate

Objective
Preparation of 1000 ml of 10X Dulbecco’s Phosphate buffered saline (PBS)

PROCEDURE

Step 1: To prepare 1000 ml of 10X PBS, weigh out 80 g NaCl (molecular weight 58.44), 2 g KCl (molecular weight 74.55), 11.5 g Na2HPO4 (molecular weight 141.96) and 2.0 g KH2PO4 (molecular weight 136.09). Transfer them to a 2 L beaker/conical flask. Add 800 ml deionized / Milli-Q water and mix it.

Tip:
One can use manual shaking using a glass rod to mix the ingredients. Magnetic stirrer makes the dissolving process easy and convenient.

Precaution:
Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute is dissolved in a solvent.

Step 2: Adjust the pH to 7.2 or 7.4 (if required) with HCl.

Step 3: Adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.

Step 4 (Optional): One can filter the solution to remove any undissolved material.

Step 5: Transfer the solution to an autoclavable bottle.

Note:
One can make small aliquots of the solution (e.g., 100 ml aliquots in 250 ml autoclavable bottles).

Step 6: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).

Note:
One can sterilize the solution by filtering through a 0.2-μm filter unit. Filter sterilization removes all suspended particles with a size of more than 0.2 μm which includes most bacteria and their spores but not mycoplasma. However, it does not inactivate enzymatic activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.

Storage
Phosphate-buffered saline (PBS) can be stored at room temperature for a few weeks. Keep the medium at 2 – 8°C for longer storage.

Note:
Storing concentrated PBS at 4°C may lead to salt precipitation. If salt is precipitated, dissolve it by keeping the solution at 37°C and by shaking it before use.

Applications

Since PBS is isotonic and has an effective pH range that corresponds to the physiological environment, it is extensively used as a physiological buffer. The most common applications of PBS are_ _ _ _
♦ In cell culture laboratories, it is extensively used as a cell washing medium.
♦ PBS is also used in immunohistochemistry/immunocytochemistry and western blot as a solvent.