How to Make a 10x Phosphate-Buffered Saline (PBS)

A 10x phosphate-buffered saline stock is made by dissolving sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate in distilled water at ten times the final working concentration, then adjusting the pH to 7.4. Keeping a concentrated stock on the shelf saves time and bench space, since you dilute it down to 1x only when you need it. The recipe itself is straightforward, but small missteps in weighing, pH adjustment, or storage can quietly ruin experiments downstream.

Reagents and Equipment

The standard 10x PBS recipe calls for four salts dissolved in one liter of ultrapure or distilled water. The amounts below yield a solution that, once diluted tenfold, gives you the classic 1x PBS used across cell culture, immunohistochemistry, and washing steps. PBS at working concentration is isotonic and sits at physiological pH, which is why it is widely regarded as biocompatible for work with living cells and tissues.¹

NaCl: 80 g
KCl: 2 g
Na₂HPO₄ (anhydrous): 14.4 g
KH₂PO₄: 2.4 g
Water: ultrapure (Type I or II) or distilled, brought to a final volume of 1 liter

You will also need a calibrated analytical balance, a magnetic stir plate with a stir bar, a graduated cylinder or volumetric flask, a pH meter with fresh calibration buffers, and either hydrochloric acid (HCl) or sodium hydroxide (NaOH) for pH adjustment. A clean glass or autoclavable bottle for storage rounds out the list.

Hydration State of Disodium Phosphate Matters

The 14.4 g figure assumes you are using anhydrous Na₂HPO₄. If your bottle contains a hydrated form, the mass needs to change because water molecules built into the crystal add weight without adding phosphate. This is one of the most common sources of error when people follow a protocol without checking the label on their reagent bottle.

Anhydrous Na₂HPO₄: 14.4 g
Na₂HPO₄·2H₂O (dihydrate): 18.0 g
Na₂HPO₄·7H₂O (heptahydrate): 26.8 g
Na₂HPO₄·12H₂O (dodecahydrate): 36.0 g

If you grab the heptahydrate off the shelf but weigh out 14.4 g as if it were anhydrous, your phosphate concentration will be roughly half what it should be. The buffer will still dissolve and look fine, but it will have less buffering capacity and the pH will drift more easily. Always check the label for the molecular formula and match your mass accordingly.

Step-by-Step Protocol

Start by adding roughly 800 mL of distilled or ultrapure water to a clean beaker or flask. You leave volume short of the final mark so you have room for pH adjustment and topping off. Drop in a magnetic stir bar and set the plate to a moderate speed.

Add NaCl first, since it makes up the bulk of the solute. Let it dissolve before adding the next salt. Then add KCl, followed by Na₂HPO₄, and finally KH₂PO₄. Each salt should dissolve fully before you add the next one. At 10x concentration the solution is noticeably saltier than 1x, so the disodium phosphate in particular can take a minute to go into solution. Gentle warming to around 30–37 °C speeds things up, but avoid heating above 50 °C because you do not need to and it creates unnecessary risk of volume loss through evaporation.

Once everything is dissolved and the solution is clear, move on to pH adjustment.

Getting the pH Right

The target pH for 10x PBS is 7.4, the same as for 1x. Some protocols call for adjusting at the 10x stage, while others recommend adjusting after dilution to 1x. Either approach works, but there is a practical wrinkle worth knowing about.

At ten times concentration, the phosphate buffer is quite strong, so the pH tends to land close to 7.4 on its own. You may only need a small amount of 1 M NaOH or 1 M HCl to nudge it into place. If your pH reads between 6.8 and 7.0 before adjustment, a few milliliters of NaOH will typically bring it up. If it reads above 7.6, a small amount of HCl brings it down. Add acid or base in small increments and let the stir bar mix for 30 seconds between additions. Overshooting and then correcting back adds unnecessary ions to the solution.

Once you hit 7.4, bring the total volume to exactly 1 liter with distilled water using a graduated cylinder or volumetric flask. Give it one final stir and check the pH once more. It should hold steady.

A note for anyone who adjusts pH after dilution instead: when you dilute 10x stock to 1x, the pH can shift slightly because dilution changes the ratio of the two phosphate species. In practice, the shift is small, rarely more than 0.1 to 0.2 pH units, but if your application is pH-sensitive it is worth checking with a meter after dilution rather than assuming the number on the stock bottle still holds.

Diluting to 1x Working Solution

To prepare 1x PBS, mix one part of 10x stock with nine parts of distilled or ultrapure water. For a common bench volume of 500 mL, that means 50 mL of 10x stock topped up to 500 mL with water. For a liter, use 100 mL of stock and 900 mL of water.

At 1x, the final ion concentrations come out to approximately 137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM phosphate buffer, and a pH of 7.4. That combination gives the solution an osmolality close to 280–300 mOsm/kg, which matches the osmolality of most mammalian body fluids. This is why 1x PBS neither swells nor shrinks cells when you wash or resuspend them.

Always use fresh, high-quality water for dilution. Tap water introduces calcium, magnesium, chlorine, and trace metals that can interfere with sensitive assays or, in the case of divalent cations, cause precipitation problems discussed below.

Why Calcium and Magnesium Are Usually Left Out

You will sometimes see recipes for “PBS with calcium and magnesium” (often labeled PBS+ or DPBS with CaCl₂ and MgCl₂). The calcium-containing version is used in specific protocols where divalent cations are needed, such as certain cell adhesion assays. But the standard 10x PBS recipe deliberately omits both, and there is a good chemical reason for that.

Calcium ions and phosphate ions have limited solubility together. When calcium is added to a phosphate-containing solution, calcium-phosphate can precipitate out as fine particles.² At 10x concentration, where the phosphate level is ten times higher than in a working solution, this precipitation risk is even greater. A cloudy stock full of invisible microcrystals is worse than useless because those particles can clog filters, scatter light in spectrophotometry, and be mistaken for biological microparticles in flow cytometry.²

If your protocol requires calcium or magnesium, the safest approach is to make a standard 10x PBS without them, dilute to 1x, and then add CaCl₂ and MgCl₂ separately from their own concentrated stocks. This keeps the phosphate concentration low enough that precipitation stays below the threshold of concern.

Storage, Shelf Life, and Sterility

A properly prepared 10x PBS stock stored in a clean, sealed bottle at room temperature will last for months. The salts are inorganic and do not degrade the way biological reagents do, so the shelf life is mainly limited by contamination. Label the bottle with the date, concentration, pH, and your initials.

For applications involving cell culture or other sterile work, you have two options. You can autoclave the 10x stock at 121 °C for 15–20 minutes, or you can filter-sterilize using a 0.22 µm membrane filter. Autoclaving is simpler but can cause trace mineral deposits on the glass over time, especially if your water quality is marginal. Filter sterilization avoids that issue and is faster for small volumes.

If you notice any cloudiness, floating particles, or a color change after storage, discard the stock and make a fresh batch. Cloudiness in PBS almost always means either microbial growth or salt precipitation from contamination with divalent cations. Neither is acceptable for experimental work.

When Sodium Azide Gets Added

Some labs add sodium azide (NaN₃) to PBS at a concentration of 0.02% to 0.1% as a bacteriostatic preservative. This is common for PBS used in antibody storage buffers or long-term immunostaining protocols, where you want to prevent microbial growth without autoclaving after every use.

Sodium azide is effective at preventing bacterial contamination, but it is not biologically inert. Research on human platelets shows that azide exerts concentration-dependent effects on cell signaling, starting at concentrations as low as 1 µmol/L.³ For antibody dilution buffers or wash solutions, those effects rarely matter. But if you are working with live cells, functional assays, or anything where subtle signaling changes could confound your results, azide-free PBS is the safer choice. Many labs keep two bottles on the shelf: one with azide for storage buffers and one without for cell-contact work.

Sodium azide is also toxic to humans and reacts with certain metals to form shock-sensitive compounds when poured down copper or lead drain pipes. Dispose of azide-containing waste according to your institution’s chemical safety guidelines, not down the sink.

Common Mistakes and How to Avoid Them

Making 10x PBS is one of the first things many researchers learn, and because the recipe looks simple, the mistakes tend to come from overconfidence rather than ignorance. A few patterns show up repeatedly.

Wrong phosphate hydration: As covered earlier, using a hydrated form of Na₂HPO₄ without adjusting the mass is the single most common formulation error. The solution will still look normal but will underperform as a buffer.
Skipping the pH check: Some people assume the salts will self-buffer to 7.4 and skip the meter entirely. Usually the pH lands close, but “close” is not good enough for pH-sensitive work. A reading of 7.0 versus 7.4 can change protein binding behavior, enzyme activity, and antibody performance.
Using tap water: Distilled or ultrapure water is not optional. Tap water varies wildly by location and can introduce calcium, iron, copper, chlorine, and organic contaminants. Even if the PBS looks fine initially, these contaminants can cause downstream problems in sensitive assays.
Topping off before adjusting pH: If you fill to the final volume first and then add NaOH or HCl, you overshoot the target volume by the amount of acid or base you added. The concentrations end up slightly off. Always adjust pH with volume to spare, then top off.
Storing at the wrong concentration: Labeling matters. If someone grabs your 10x stock thinking it is 1x and uses it to wash cells, the osmolality will be roughly ten times physiological. Cells will shrink dramatically and likely die. Clearly marking the bottle “10x” in large text prevents this.

How PBS Interacts with Lipid Structures

If you work with liposomes, vesicles, or lipid nanoparticles, PBS is not quite the inert bystander it appears to be in most protocols. Research on phosphatidylcholine vesicles shows that PBS, depending on how it is added, can alter vesicle structure. When PBS is used to induce osmotic shock, for example, vesicles can elongate and change shape rather than simply sitting passively in solution.⁴ The method of addition matters too: dumping concentrated PBS into a vesicle suspension creates a sudden osmotic gradient that affects morphology differently than a slow, gradual addition.

For most routine lab work, this is irrelevant. But for anyone developing lipid-based drug delivery systems, studying membrane biophysics, or using vesicles as model membranes, the takeaway is that PBS concentration and how you introduce it to your sample are variables worth controlling. Diluting your 10x stock to 1x before it touches lipid preparations, and adding it slowly with mixing, minimizes osmotic artifacts.

Variations on the Standard Recipe

The recipe given above is the most widely used formulation, sometimes called Dulbecco’s PBS (DPBS) without calcium and magnesium. But you will encounter minor variations depending on the source. Some older protocols use slightly different phosphate ratios, and a few call for Na₂HPO₄ at 11.5 g per liter of 10x with a correspondingly higher amount of NaH₂PO₄ (monosodium phosphate) instead of KH₂PO₄. These tweaks shift the potassium content and can change the exact pH landing point before adjustment.

In practice, the differences between these formulations are small enough that they rarely matter for routine washing, blocking, and dilution steps. Where they can matter is in electrophysiology, certain enzyme kinetics experiments, and any assay where the exact potassium concentration affects the readout. If a published protocol specifies a particular PBS formulation by name, match it rather than substituting a generic recipe.

Another variation you may see is Tris-buffered saline (TBS), which replaces the phosphate buffer with Tris. TBS is preferred for applications where phosphate ions interfere, such as alkaline phosphatase detection in Western blots or any assay that uses phosphate as a substrate or product. If your protocol says “use TBS, not PBS,” there is a reason, and swapping in PBS will give you high background or false signals.

Buying Premade Versus Making Your Own

Commercial 10x PBS is available from every major lab supplier in volumes ranging from 500 mL to 20 liters. It comes with a certificate of analysis listing the exact pH, osmolality, endotoxin level, and sterility assurance. For cell culture core facilities or labs that go through large amounts of PBS daily, buying premade can be worth the cost simply to save technician time and reduce batch-to-batch variability.

The tradeoff is price. A liter of 10x PBS from a supplier might cost several times more than the raw salts needed to make the same volume. For a lab that uses PBS primarily for non-sterile washing steps in immunohistochemistry or ELISA, making it in-house is cheaper and perfectly adequate. The salts are stable, inexpensive, and available in large quantities.

A middle-ground approach that many labs use is to buy commercial 10x PBS for anything touching live cells and make their own for wash buffers and general bench use. This way, the sterility-critical applications get a quality-controlled product, while the high-volume, low-stakes applications run on the cheap homemade version. Just be sure to label each bottle clearly so nobody reaches for the wrong one.