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Preparation of 10X Phosphate buffered saline (DPBS), Dulbecco's method

Overview
  • Phosphate-buffered saline (PBS) is a buffer solution commonly used in biological research.

  • PBS is isotonic and non-toxic to cells. It is used in several applications of tissue culture, most notably for cell washing and trypsinization. These applications are dependent on the ability of PBS to maintain the cells in a viable and healthy state for short duration.

  • PBS supports the cell viability by keeping the medium pH and osmolality near physiological.

  • Several formulations of PBS have been described. Most formulations contain similar amount of sodium chloride and potassium chloride, but differ in concentration of Na2HPO4 and KH2PO4. PBS can also be supplemented with Calcium chloride and Magnesium chloride. Some of the PBS formulations are_ _ _ _ _ _

    • PBS : 137 mM Sodium chloride, 2.7 mM Potassium chloride, 10 mM Disodium hydrogen phosphate, 1.8 mM Potassium dihydrogen phosphate

    • Dulbecco's PBS : 137 mM Sodium chloride, 2.7 mM Potassium chloride, 8.1 mM Disodium hydrogen phosphate, 1.47 mM Potassium dihydrogen phosphate

  • PBS, used for cell culture applications, often do not contain calcium chloride and magnesium chloride but may contain glucose or pyruvate. Divalent cations inhibit trypsinization reaction (for subculturing). Glucose or pyruvate serve as energy source and is added in PBS to keep the cells alive and healthy for relative longer time.

  • PBS, used for immunocytochemistry/immunohistochemistry, is supplemented with divalent alkaline earth metals (calcium chloride and magnesium chloride). Divalent alkaline earth metals is helpful to ensure that uncondensed chromatin remains intact and contained within the nucleus during the staining procedure. It also dramatically decreases background fluorescence levels when using DNA probes excited by ultraviolet irradiation (DAPI and Hoechst).

Phosphate buffered saline | Preparation of 10X Phosphate buffer saline (PBS) | Preparation of 10X Dulbecco's Phosphate buffered saline (DPBS) containing calcium chloride and magnesium chloride |
 
Requirement
  • Reagents
    • Sodium chloride (NaCl)
    • Potassium chloride (KCl)
    • Disodium hydrogen phosphate, anhydrous (Na2HPO4)
    • Potassium dihydrogen phosphate (KH2PO4)
    • Deionized / Milli-Q water

  • Equipments and disposables
    • Measuring cylinder
    • 2 litre Conical flask / Beaker
    • pH meter and/or pH paper
    • Magnetic stirrer with heat control (optional)
 
Composition of 10X Phosphate buffer 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 Phosphate buffer saline (PBS) by Dulbecco's method
    This protocol is adapted from the following article.
    Dulbecco, R. and Vogt, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med. 99 (2), 167-182.
    Pubmed link | Full text from PubmedCentral

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Preparation

  • To prepare 1000 ml of 10X PBS, weigh 80 gram NaCl (molecular weight 58.44), 2 gram KCl (molecular weight 74.55), 11.5 gram Na2HPO4 (molecular weight 141.96) and 2.0 gram KH2PO4 (molecular weight 136.09). Transfer them to 2 litre beaker/conical flask. Add 800 ml deionized / Milli-Q water and mix it.
    Tip(s):
    One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the dissolving process easy and convenient.
    Precaution(s):
    Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute is dissolved in solvent. see solution preparation

  • Adjust the pH to 7.2 or 7.4 (if required) with HCl.
    Precaution(s):
    Since pH is dependent on temperature, It is important to adjust the medium pH at room temperature (25°C).

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

  • Optional : One can filter the solution to remove any undissolved material.

  • Transfer the solution to autoclavable bottle.
    Note(s):
    One can make small aliquots of the solution (e.g., 100 ml aliquots in 250 ml autoclavable bottle).

  • Sterilize the solution by autocaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).
    Note(s):
    One can sterilize the solution by passing through 0.2μ filter unit. Filter strilization removes all suspended particles with size more than 0.2 μ which includes most bacteria their spores but not mycoplasma. Morover, it does not inactivate enzymatic activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorgasnisms including mycoplasma.


Storage
  • Phosphate-buffered saline (PBS) can be stored at room temperature for few weeks. Keep the medium at 2 - 8°C for longer storage.
    Note(s):
    Storing concentrated DPBS at 4°C may lead to salt precipitation. If salt is precipitated, dissolve it by keep solution at 37°C and by shaking before use.

 
Applications
  • Since PBS is isotonic and has effective pH range which correspond to physiological environment, it is extensively used as a physiological buffer. The most common applications of PBS are

    • In cell culture laboratory, it is extensively used as a cell washing medium.

    • PBS is also used in immunohistochemistry/immunocytochemistry and western blot as a solvent.

Preparation of 10X 1000 ml Dulbecco'S Phosphate Buffered Saline (PBS)
To prepare Phosphate Buffered Saline of various volume (100 ml, 500 ml, 1,000 ml and 10,000 ml), follow the table.
Reagents / Volume 100 ml 500 ml 1,000 ml 10,000 ml
NaCl 8 g 40 g 80 g 800 g
KCl 0.2 g 1 g 2 g 20 g
Na2HPO4 1.15 g 5.75 g 11.5 g 115 g
KH2PO4 0.20 g 1.0 g 2.0 g 20 g
Deionized / Milli-Q water Adjust to 100 ml Adjust to 500 ml Adjust to 1000 ml Adjust to 10,000 ml


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