PBS vs DPBS: What Is Different, and Which One to Use

Phosphate-buffered saline is an isotonic salt solution buffered near physiological pH by a monobasic/dibasic phosphate pair. It contains no glucose, no amino acids, no protein and no bicarbonate. It is a handling solution - for rinsing monolayers, diluting cells, carrying cells between steps and washing away serum - not a medium in which cells can be kept.

The phosphate pair is what makes it useful outside an incubator. The second dissociation of phosphoric acid has a pKa of about 7.2, almost exactly the pH at which cells are cultured, so a phosphate buffer holds pH well in room air. Bicarbonate-buffered media rely on the carbonic acid system, whose relevant pKa is about 6.1, and therefore need a CO2 atmosphere to hold pH 7.4. This is the reason a bench-top wash step is done in PBS rather than in medium.

DPBS is Dulbecco's phosphate-buffered saline, from the formulation Renato Dulbecco and Marguerite Vogt described in 1954 while developing plaque assays for animal viruses. "DPBS" therefore names a specific historical recipe. "PBS" names a class of solutions, and different suppliers sell meaningfully different ones under that name.

Comparing the two Gibco products that carry these exact names makes the difference concrete.

Gibco PBS pH 7.4 contains sodium chloride at 9.0 g/L (155 mM), potassium phosphate monobasic at 144 mg/L (1.06 mM) and sodium phosphate dibasic heptahydrate at 795 mg/L (2.97 mM). Total phosphate is about 4.0 mM, and there is no potassium chloride at all.

Gibco DPBS without calcium and magnesium contains sodium chloride at 8.0 g/L (137.9 mM), potassium chloride at 200 mg/L (2.67 mM), potassium phosphate monobasic at 200 mg/L (1.47 mM) and sodium phosphate dibasic heptahydrate at 2160 mg/L (8.06 mM). Total phosphate is about 9.5 mM.

So DPBS has less sodium chloride, adds potassium chloride, and carries roughly 2.4 times the phosphate. The practical consequence is buffering capacity: at about 9.5 mM phosphate, DPBS resists a pH shift considerably better than a 4 mM PBS when you add cells, acidic reagents or a slug of medium to it. If you are holding a cell suspension on the bench for twenty minutes, that difference is real.

One caution about the word PBS. The recipe most molecular biology labs make from powder or from a Sambrook-style protocol - 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4 - is close to Dulbecco's formulation, with about 12 mM total phosphate. That home-made "PBS" is nearer to DPBS than it is to a commercial bottle labelled PBS pH 7.4. Always read the formulation on the label rather than inferring composition from the three or four letters on the front.

Both PBS and DPBS are sold in a version containing calcium and magnesium and a version without. This is the choice that changes experimental outcomes, and it is independent of whether the bottle says PBS or DPBS.

Gibco DPBS with calcium and magnesium adds calcium chloride at 100 mg/L (0.90 mM Ca2+) and magnesium chloride hexahydrate at 100 mg/L (0.49 mM Mg2+) to the same base. Nothing else changes.

Both cations are structural to cell adhesion. Cadherins - the proteins that hold cells to each other at adherens junctions - are calcium-dependent by definition; their extracellular domains require bound Ca2+ to adopt the rigid conformation that permits binding, and they lose adhesive function and become protease-sensitive when calcium is removed. Integrins, which attach cells to the extracellular matrix, coordinate a divalent cation at the metal-ion-dependent adhesion site in their ligand-binding pocket and do not bind ligand without it.

Remove Ca2+ and Mg2+ and you destabilise both systems at once. Add them and you preserve both. That is the whole decision.

The most common error with these solutions is washing a monolayer with a calcium- and magnesium-containing solution immediately before adding trypsin, and then wondering why the cells take twelve minutes to lift and come off in sheets.

The wash before trypsinisation has two jobs. The obvious one is removing serum, because serum contains alpha-1-antitrypsin and other protease inhibitors that will neutralise the trypsin you are about to add. The less obvious one is stripping divalent cations out of the junctions, so that the cadherin contacts are already loosened when the enzyme arrives. A Ca/Mg-containing wash does the first job and actively undoes the second.

The result is a longer digestion. Longer digestion means more proteolysis of the surface proteome, lower viability, and more of the clumping that spoils a haemocytometer count or a single-cell suspension for flow cytometry or sequencing. Standard trypsin-EDTA solutions are themselves formulated in a calcium- and magnesium-free balanced salt solution and contain EDTA precisely to chelate residual cations, so a Ca/Mg wash means the EDTA in your dissociation reagent is spent on undoing your own wash step.

The converse error is real too. If you wash an adherent monolayer repeatedly in a Ca/Mg-free solution when you intend the cells to stay put - during an immunofluorescence protocol, before fixation, or between stimulation steps - you will lose cells from the plate and see patchy, retracted morphology. For those steps, the calcium- and magnesium-containing version is the correct choice.

Hanks' Balanced Salt Solution is the third bottle in this argument, and it differs from both phosphate salines in two ways that decide when to reach for it.

First, HBSS contains D-glucose at 1.0 g/L (5.6 mM). PBS and DPBS contain no energy source whatsoever. Any procedure in which cells sit in the solution for more than a few minutes at 37 C - tissue dissociation, organ perfusion, a long sort, imaging on a warmed stage - should use HBSS or EBSS rather than PBS.

Second, HBSS is not primarily a phosphate buffer. It carries only about 0.78 mM total phosphate (potassium phosphate monobasic 60 mg/L plus sodium phosphate dibasic 48 mg/L) and instead uses sodium bicarbonate at 350 mg/L (4.17 mM). That low bicarbonate level is deliberate: Hanks' salts are formulated to hold pH in ambient air, in a closed vessel, without a CO2 incubator.

Earle's Balanced Salt Solution is the opposite design. It carries sodium bicarbonate at 2.2 g/L (26.2 mM) and is intended for use in a 5% CO2 atmosphere. Put EBSS on the open bench and it will drift alkaline as CO2 leaves; put a Hanks'-based medium in a 10% CO2 incubator and it will go acid. The general rule suppliers give is to match bicarbonate to atmosphere: below about 1.5 g/L for ambient or 4% CO2, 1.5-2.2 g/L for 5%, 2.2-3.4 g/L for 7%, and above 3.5 g/L for 10%.

HBSS and EBSS also both contain calcium and magnesium in their standard form, and both are sold Ca/Mg-free for dissociation work, exactly as the phosphate salines are.

Use a calcium- and magnesium-free PBS or DPBS for: rinsing serum off a monolayer before trypsin, TrypLE or EDTA dissociation; diluting or resuspending cells for counting; washing pellets before lysis; preparing single-cell suspensions for flow cytometry or single-cell sequencing; and as the diluent for EDTA-based dissociation solutions.

Use a PBS or DPBS containing calcium and magnesium for: washing adherent cells you intend to keep adherent; wash steps in immunocytochemistry and immunofluorescence; assays where divalent cations are functionally required, such as integrin- or lectin-mediated binding; and resuspending cells that must retain surface adhesion competence.

Use HBSS or EBSS instead of either when: cells will be in the solution longer than roughly fifteen minutes at 37 C, or the procedure needs an energy source - primary tissue dissociation, perfusion, transport of biopsies, extended live-cell handling. Choose Hanks' salts for work in air and Earle's salts for work in a CO2 incubator.

Do not use any of them as a medium. None of these solutions contains amino acids, vitamins or protein. Cells held in balanced salt solution deplete, and viability falls measurably over an hour or two even in the glucose-containing versions.

A bottle labelled DPBS is not necessarily at pH 7.4. Gibco's DPBS specifications are pH 7.0-7.3 without calcium and magnesium and pH 7.0-7.2 with, while the product sold as PBS pH 7.4 is titrated to 7.4. If your protocol depends on a starting pH - a pH-sensitive dye, a fluorophore with a pH-dependent quantum yield, a binding assay - check the certificate of analysis instead of assuming.

Concentrates and powders are usually Ca/Mg-free. Calcium and magnesium phosphates have limited solubility, so 10X concentrates, powdered formats and anything intended to be autoclaved are generally supplied without divalent cations, which are added afterwards from a separate sterile stock. If you dilute a 10X DPBS and expect calcium in it, check the label.

Autoclaving Ca/Mg-containing phosphate saline can precipitate it. Sterile filtration at 0.2 um is the safer route for any phosphate saline containing calcium.

Phenol red is optional and irrelevant to the buffering. Most PBS and DPBS is supplied without it. Versions with phenol red exist mainly so you can see gross pH drift or contamination; they will interfere with absorbance and fluorescence readings in the same way phenol red in medium does.

PBS-EDTA is a different product. A PBS or DPBS containing 0.5 mM EDTA is a dissociation reagent, not a wash buffer. Do not substitute it for plain PBS in a rinse step, and do not leave cells in it while you attend to something else.