HBSS (Hank's Balanced Salt Solution)

HBSS is a balanced salt solution, not a growth medium. It supplies the inorganic ions a cell needs to hold its osmotic balance and membrane potential, plus glucose as an immediate energy source, but no amino acids, no vitamins and no protein. Cells survive in it for hours; they do not grow in it.

The formulation traces to John Hanks, who developed the salt mixture in the 1940s; the commonly cited paper is Hanks and Wallace, 1949, on preserving tissues by refrigeration. The design intent shows in the composition: Hanks' salts were meant for handling and transporting tissue under ordinary atmospheric conditions, so the bicarbonate is low and the phosphate does most of the buffering.

The everyday uses are:

• Washing cells and monolayers before trypsinisation, fixation or lysis, to remove serum and residual medium
• Rinsing between staining or assay steps
• Transport and short-term holding of tissue and primary isolates
• Diluting enzymes, reagents and cell suspensions
• Assay buffer for short-duration functional work, where glucose but not full medium is wanted

HBSS is deliberately simple, and its simplicity is why it works: nothing in it interferes with a downstream enzymatic step or a protein assay.

The standard Hanks' balanced salt solution formulation, in both supplied forms:

The two forms are otherwise identical — only the three divalent cation salts are removed. Total magnesium in the with-Ca/Mg form is about 0.9 mM, split between the sulfate and chloride salts; calcium is 1.26 mM. These are standard formulation values and are the same across suppliers, since Hanks' salts is a published standard rather than a proprietary formulation.

Two features define how HBSS behaves.

The bicarbonate is very low — 350 mg/L, against 2,000 mg/L in RPMI 1640 and 3,700 mg/L in DMEM. Hanks' salts were formulated for atmospheric CO2. Most of the pH stability in HBSS comes from the phosphate pair (KH2PO4 and Na2HPO4), which buffers independently of CO2.

It contains glucose at 5.55 mM, unlike PBS and DPBS. This is a real functional difference: cells held in HBSS have an energy source, which matters during long dissociations, tissue transport and functional assays. It also means HBSS supports bacterial growth if it becomes contaminated, so treat it with the same aseptic care as medium.

Total osmolality is close to physiological, around 280–300 mOsm/kg, and the working pH is about 7.0–7.4.

This is the choice that most often goes wrong, and the logic is straightforward once stated.

Divalent cations are required for cell adhesion. Calcium is essential to cadherin-mediated cell–cell junctions; calcium and magnesium together are required for integrin-mediated attachment to the substrate. Remove them and adhesion weakens.

Use calcium- and magnesium-free HBSS when you want cells to detach. This is the wash you use immediately before trypsin or another dissociation reagent. Rinsing a monolayer with Ca/Mg-free HBSS both removes serum — which contains protease inhibitors that would blunt the trypsin — and begins loosening junctions, so the subsequent dissociation is faster and gentler. Ca/Mg-free HBSS is also the correct diluent for EDTA-based dissociation reagents, since calcium in the buffer would simply chelate the EDTA.

Use HBSS with calcium and magnesium when you want cells to stay attached and functional. Washing a monolayer you intend to keep, holding a primary isolate, transporting tissue, and any functional assay where signalling matters — calcium is a second messenger, and a calcium-free buffer will distort responses in imaging, secretion and activation assays. Divalent cations are also required for many enzymes, including collagenase, so collagenase digestions should use the Ca/Mg-containing form.

A useful rule of thumb: if the next step involves an enzyme or a live functional measurement, use with-Ca/Mg. If the next step is detachment, use Ca/Mg-free.

These three are used almost interchangeably in practice, and mostly that is fine — but not always. The comparison table on this page gives the component-level view; the practical distinctions are these.

Glucose. HBSS has it (5.55 mM), PBS and DPBS do not. If cells will be held for more than a few minutes, or are being dissociated, transported, or measured functionally, HBSS's glucose is a genuine advantage. For a 30-second rinse before lysis it makes no difference.

Buffering. DPBS is the strongest simple buffer of the three — its phosphate is around 8 mM total, several times HBSS's roughly 0.8 mM — and it holds pH well on the open bench with no CO2. HBSS relies on a weaker phosphate buffer plus a small bicarbonate contribution. In a 5% CO2 incubator, HBSS's bicarbonate is far below what would be needed to hold pH, so HBSS is not appropriate for extended incubator use.

Divalent cations. All three are supplied with and without calcium and magnesium, and the same selection logic applies to all three.

Ionic detail. HBSS is closer to a medium's ionic profile — it carries magnesium as both sulfate and chloride and has a lower phosphate load. DPBS is a saline with a heavy phosphate buffer. That heavy phosphate is a liability in one specific case: phosphate precipitates calcium, so DPBS with calcium can form insoluble calcium phosphate on long storage or after autoclaving, and phosphate interferes with some phosphate-sensitive assays and with mineralisation studies.

Choosing quickly: rinsing before lysis or fixation, any of the three. Before trypsinisation, Ca/Mg-free — any of the three, but HBSS or DPBS conventionally. Tissue transport or long dissociation, HBSS with Ca/Mg for the glucose. Long bench-top steps with no CO2 and no cells to feed, DPBS for the stronger buffer. Mineralisation or phosphate-sensitive assays, HBSS rather than DPBS.

1X HBSS is used at pH 7.0–7.4. Manufacturers' release specifications for the ready-to-use 1X solution are typically set around 7.0–7.2, with some products carrying a wider tolerance, and the solution is functionally correct anywhere in the 7.0–7.4 band for washing and short-term holding.

10X HBSS concentrate is deliberately acidic, usually around pH 5.8–6.1. This is not a defect and does not need correcting in the bottle. At ten times concentration the calcium, magnesium, phosphate and bicarbonate salts would precipitate at neutral pH, so the concentrate is held acidic to keep them in solution. The pH comes up to the normal range on dilution to 1X. If you have measured a 10X stock, found it at pH 6, and are wondering whether it has gone off — it has not.

HBSS is buffered principally by its phosphate pair, with a small contribution from 4.17 mM bicarbonate. Phosphate buffers well around pH 7.2 and is independent of CO2, which is exactly what a bench-top handling solution needs.

The consequence is that HBSS is not an incubator solution. In a 5% CO2 atmosphere, dissolved CO2 acidifies the solution and 4 mM bicarbonate cannot hold against it. Cells left in HBSS in a CO2 incubator sit at a falling pH — one of those errors that produces slow, unexplained loss of viability rather than an obvious failure.

Conversely, HBSS left standing at atmospheric CO2 slowly loses its small bicarbonate as CO2 and drifts alkaline. In practice this is minor and does not matter over the time scales HBSS is normally used for.

If you need a solution that holds pH reliably during a long procedure outside the incubator, HBSS buffered with 10–25 mM HEPES is the usual answer. HEPES buffers well at pH 7.2–7.4 without CO2. The trade-offs are the same as always: HEPES generates hydrogen peroxide under light and is cytotoxic to some primary cells at 25 mM.

Storage. HBSS is stable at 2–8 °C, or at room temperature for some formulations — follow the supplier's label. Unopened shelf life is typically 12–24 months. Because it contains glucose, it will support microbial growth if contaminated; work aseptically and do not decant back into the stock bottle.

Temperature. For washing adherent cells, use HBSS at room temperature or 37 °C. Ice-cold buffer on a warm monolayer causes cold shock and can trigger detachment or membrane damage. Cold HBSS is appropriate when you deliberately want to slow metabolism — tissue transport, some primary isolations — but that is a decision, not a default.

Do not use HBSS as a culture medium. It has no amino acids, vitamins, lipids or protein. Cells tolerate a few hours; overnight in HBSS is a viability experiment, not a culture.

Common mistakes:

• Using Ca/Mg-containing HBSS to rinse before trypsinisation. The divalent cations maintain adhesion and the dissociation takes longer and needs more enzyme, increasing damage.
• Using Ca/Mg-free HBSS as a collagenase diluent. Collagenase requires calcium; the digestion will underperform.
• Diluting EDTA dissociation reagents in Ca/Mg-containing buffer. The calcium consumes the chelator.
• Leaving cells in HBSS in a CO2 incubator. Its bicarbonate cannot buffer at 5% CO2 and the pH falls.
• Assuming HBSS and PBS are equivalent for functional assays. The glucose and the different ionic balance matter whenever cells are being measured rather than merely rinsed.
• Using DPBS for mineralisation work. Its high phosphate confounds calcium phosphate deposition; HBSS is the better choice.