Sodium chloride in cell-culture media and physiological buffers

Sodium chloride (NaCl, CAS 7647-14-5, EC 231-598-3, M = 58.44 g/mol) is the largest single contributor to the osmolality of cell-culture media and physiological buffers. It supplies most of the Na⁺ and Cl⁻ that cells need to hold their volume and membrane potential; sodium bicarbonate, sodium phosphate and other salts supply the rest of the sodium.

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How much NaCl do common media and buffers contain?

NaCl content of classical formulations, per litre of 1× medium:

• DMEM: 6.4 g/L (110 mM)
• RPMI-1640: 6.0 g/L (103 mM)
• MEM (Earle's salts): 6.8 g/L (116 mM)
• Ham's F-12: 7.6 g/L (130 mM)
• PBS (Dulbecco's, 1×): 8.0 g/L (137 mM)
• HBSS: 8.0 g/L (137 mM)
• 0.9 % saline: 9.0 g/L (154 mM)

Media contain less NaCl than PBS because sodium bicarbonate (3.7 g/L, 44 mM in DMEM; 2.0 g/L, 24 mM in RPMI-1640) and sodium phosphate add further sodium and osmolality. Total Na⁺ in most media is therefore around 135–155 mM, close to the 135–145 mM of human plasma. Manufacturers' variants (e.g. different glucose or HEPES content) can differ, so check the formulation sheet of the product you actually use.

Why does osmolality matter?

Most mammalian cell lines grow well between roughly 260 and 340 mOsm/kg, and most media are formulated at 280–320 mOsm/kg; human plasma is 275–295 mOsm/kg. Each millimole of NaCl per litre adds just under 2 mOsm/kg. In a hypertonic medium water leaves the cells and they shrink; in a hypotonic medium they swell and, if the difference is large enough, lyse.

Because NaCl is the biggest contributor, it is the component usually adjusted when osmolality has to be corrected — for example after adding HEPES, supplements or drugs dissolved in water. Measure the result with an osmometer rather than relying on a calculation, and remember that evaporation in an incubator raises osmolality over time.

What does NaCl do in buffers such as PBS?

NaCl does not buffer pH — in PBS that is the job of the phosphate. NaCl sets the tonicity and most of the ionic strength (about 0.17 M in 1× PBS). Ionic strength affects protein–protein and antibody–antigen interactions, which is why wash and dilution buffers for ELISA and Western blotting are usually PBS or TBS with 137–150 mM NaCl. Some protein purifications use higher salt, such as 300 mM NaCl in nickel-affinity lysis buffers, to suppress non-specific binding.

Which grade should you use?

For cell culture and buffer preparation, use a grade with a defined specification and a lot-specific Certificate of Analysis (CoA):

• Pharmacopoeial grades (Ph. Eur., USP, JP): assay 99.0–100.5 % on the dried substance, with limits for impurities such as sulfate, bromide, iodide, iron and barium. Material for GMP manufacture is normally pharmacopoeial grade from a qualified supplier.
• ACS reagent grade: assay at least 99.0 % with specified impurity limits. Suitable for most laboratory buffers, but usually not tested for endotoxin.
• Cell-culture-tested or low-endotoxin grades: add endotoxin testing (LAL method, Ph. Eur. 2.6.14 / USP <85>) and sometimes a cell-growth test. Limits are set by each supplier, so compare the specification rather than the grade name.

Table salt is not suitable: it contains anti-caking agents and often added iodide or iodate.

Preparation and storage

NaCl is chemically stable. Keep the container tightly closed in a dry place at room temperature, because the solid absorbs moisture and cakes at high humidity. Prepare solutions with purified laboratory water (type I or equivalent); NaCl solutions can be sterilised by autoclaving or by 0.22 µm filtration.

Sodium chloride is registered under REACH, listed on the US TSCA Inventory and not classified as hazardous under GHS/CLP. Those rules govern supply and safety information; whether a lot is suitable for cell culture is shown by its grade specification and CoA.

Frequently asked

• How much NaCl is in 1× PBS? 8.0 g/L, i.e. 137 mM (Dulbecco's formulation), together with 2.7 mM KCl and roughly 10 mM phosphate.

• Why does DMEM contain less NaCl than PBS? Sodium bicarbonate (3.7 g/L in DMEM) and sodium phosphate supply additional sodium and osmolality, so less NaCl is needed. Total Na⁺ in DMEM is about 155 mM.

• What grade of NaCl should I use for cell culture? A pharmacopoeial (Ph. Eur./USP) or cell-culture-tested grade with a lot-specific CoA. If endotoxin matters, choose a grade that is endotoxin-tested; ACS reagent grade usually is not.

• Can I use table salt? No. Table salt contains anti-caking agents (often ferrocyanides) and frequently added iodide or iodate, and its impurity profile is not specified.