warning For Research Use Only (RUO). Not intended for clinical, diagnostic, or therapeutic use in humans.
verified Gas Phase · Divalent-Cation Selection Framework
Balanced Salt Solutions — A Selection Guide for the Research Laboratory
Four formulations, one page. Pick the salt solution your workflow needs and go straight to its product page — the composition tables, selection logic, and handling guidance follow below.
Balanced Salt Solutions — Catalog · 4 Formulations
Select the solution that matches your workflow — click View for the product page.
Balanced Salt Solution
Buffering & Cations
Best For
Product Page
Hank's Balanced Salt Solutions
Ambient air · HCO₃? low (~4 mM) · Ca²?/Mg²? with or without
What a balanced salt solution is — and what it is not
A balanced salt solution (BSS) is a buffered, isotonic salt solution that holds cells and tissues at physiological pH, osmolality, and ionic balance during handling. It is not a growth medium. It carries no amino acids, vitamins, or serum, so it is used for washing, dissociation prep, dilution, transport, dissection, and assay setup, not for proliferation in the incubator.
The parameters that decide which formulation fits a protocol — and the ones a single product name will not tell you.
Four formulations stocked: Hank's (HBSS), Earle's (EBSS), Tyrode's, and Spinner Balanced Salts (Eagle, Ca²?-free).
Gas phase drives the EBSS vs HBSS split: EBSS is buffered for 5% CO₂; HBSS is buffered for ambient air. Match the solution to the gas phase of the work, not to the cell line.
Bicarbonate content is the mechanism: EBSS carries high bicarbonate (~26 mM), HBSS low (~4 mM), Tyrode's moderate.
Divalent cations act as an adhesion switch: Ca²? and Mg²? maintain cadherin (cell–cell) and integrin (cell–substrate) adhesion.
Calcium/magnesium-free (CMF) forms prime cultures for enzymatic dissociation, usually with trypsin and EDTA.
Spinner salts omit Ca²? specifically to prevent aggregation in suspension.
Every formulation carries D-glucose at 1000 mg/L, and all are isotonic and pH-buffered — but none contains amino acids, vitamins, or serum.
Phenol red is not inert: it has weak estrogenic activity and overlaps common fluorescence channels.
BUFFERED · ISOTONICNOT A GROWTH MEDIUMRUO
CATEGORY REFERENCE · BALANCED SALT SOLUTIONS
Selection at a glance — buffering, divalent cations, and representative composition (mg/L)
Gas phase and divalent cations. Get either one wrong and the solution works against the protocol — drifting pH in one direction, or holding cells together when you need them apart.
air
Gas phase drives the EBSS vs HBSS split
EBSS is buffered for 5% CO₂; on the open bench it outgasses CO₂ within minutes and drifts alkaline. HBSS is buffered for ambient air; in a CO₂ incubator it over-acidifies. Match the solution to the gas phase of the work, not to the cell line.
tune
CO₂-independent buffering on the bench
Need CO₂-independent buffering on the bench? Add HEPES at 10 to 25 mM — but note it can be phototoxic under light and cytotoxic at higher concentrations.
link
Divalent cations act as an adhesion switch
Ca²? and Mg²? maintain cadherin (cell–cell) and integrin (cell–substrate) adhesion. Keep them when adhesion, spreading, or junction integrity must be preserved.
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Remove them to dissociate
Remove Ca²? and Mg²? (calcium/magnesium-free, CMF) to prime cultures for enzymatic dissociation, usually with trypsin and EDTA. Rule of thumb: cells staying attached, keep Ca²?/Mg²?; cells being detached, remove both.
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Suspension work omits calcium
Spinner salts omit Ca²? specifically to prevent aggregation in suspension, which is why the Eagle spinner formulation is supplied without calcium chloride for suspension and spinner-flask culture.
visibility
Phenol red is not inert
It has weak estrogenic activity[1] and overlaps common fluorescence channels. Use phenol-red-free BSS for imaging, flow, and hormone-response assays.
The mistake that costs a plate
Choosing the solution by cell line instead of by gas phase. The two workhorses differ roughly six-fold in bicarbonate: EBSS is built for the 5% CO₂ incubator, HBSS for the open bench. Used in the wrong atmosphere, EBSS drifts alkaline within minutes of leaving the incubator and HBSS over-acidifies inside it.
~26
mM bicarbonate in EBSS — buffered for a 5% CO₂ incubator
~4
mM bicarbonate in HBSS — buffered for ambient air
History & Standardization
How the named salt solutions became standard
Each formulation on this page carries the name of the laboratory that published it, and each reference below explains a parameter you still specify when ordering. This chronology is built from the source's own reference list.
1
1943 Earle — the CO₂-buffered salt base
Earle WR, Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J Natl Cancer Inst 4:165–212. The salt solution described in this line of work is the one still supplied as Earle's Balanced Salt Solution (EBSS), buffered with high bicarbonate for a 5% CO₂ environment.
2
1949 Hanks & Wallace — the ambient-air salt base
Hanks JH, Wallace RE, Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc Soc Exp Biol Med 71:196–200. The low-bicarbonate solution from this work is supplied today as Hanks' Balanced Salt Solution (HBSS), buffered for work on the open bench.
3
1970 Waymouth — osmolality becomes a specification
Waymouth C, Osmolality of mammalian blood and of media for culture of mammalian cells. In Vitro 6:109–127. Isotonicity stopped being an assumption and became a measured, matched parameter — one of the three things a BSS is required to hold, alongside physiological pH and ionic balance.
4
1986 Berthois et al. — phenol red is not inert
Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS, Phenol red in tissue culture media is a weak estrogen. PNAS 83:2496–2500. The pH indicator itself turned out to be biologically active, which is why phenol-red-free formats exist for imaging, flow, and hormone-response work.
5
2015 Freshney — consolidated bench practice
Freshney RI, Culture of Animal Cells, 7th ed., Wiley-Blackwell. The selection logic on this page — gas phase, divalent cations, warming, pH confirmation — is the practice codified in the standard manual.
Selection & Composition Reference
Selecting a BSS, and what is actually in it
The first table is the selection matrix; the second gives representative composition in mg/L. Use them together — buffering decides where the solution can be used, composition decides what it will do to the cells.
Table 1. Selecting a BSS
Solution
Buffered for
Bicarbonate
Ca²? / Mg²?
Glucose
Best for
Earle's (EBSS)
5% CO₂ incubator
High (~26 mM)
Present
Yes
CO₂-environment handling, metabolic assays, media base
Organ/tissue perfusion, GI and cardiac prep, platelet aggregometry
Spinner salts (Eagle)
5% CO₂ incubator
High
Ca²? omitted
Yes
Suspension and spinner-flask culture (prevents clumping)
Dulbecco's PBS (DPBS)*
Ambient air
None (phosphate)
With or without
Optional
General washing and rinsing; CMF form for dissociation
*DPBS is phosphate-buffered rather than a true bicarbonate BSS, but it is used interchangeably for many wash steps.
Table 2. Representative composition (mg/L)
Approximate values for orientation. Exact figures and salt hydration forms vary by supplier; verify against the specific SKU.
Component
EBSS
HBSS
Tyrode's
NaCl
6800
8000
8000
KCl
400
400
200
CaCl₂ (anhyd.)
200
140
200
MgSO₄ (anhyd.)
98
98
—
MgCl₂ (anhyd.)
—
48
100
NaHCO₃
2200
350
1000
Na phosphate (mono/dibasic)
122
48 + 60
50
D-Glucose
1000
1000
1000
Phenol red
10
11
—
Read Table 2 as the complete formulation. The composition column for each solution is the full, divalent-cation-containing form. Where Table 1 lists Ca²?/Mg²? as “with or without,” the calcium/magnesium-free (CMF) version of that solution is the same formulation with the CaCl₂, MgSO₄, and MgCl₂ rows omitted — which is exactly what makes it suitable for dissociation prep. Since exact figures and salt hydration forms vary by supplier, verify against the specific SKU before matching a composition to a protocol.
Applications by Solution
Which solution for which bench workflow
Select a formulation to see the workflows it is intended to support.
Ambient air · low bicarbonate (~4 mM) · with or without Ca²?/Mg²?
Open-bench rinses
Resuspension
Transport
Dissociation prep (CMF form)
Rinsing serum before trypsinization (CMF HBSS — serum inhibits trypsin, so clear it first)
Priming cells for dissociation (CMF HBSS + EDTA)
Flow cytometry / live-cell imaging buffer base (phenol-red-free HBSS)
Ambient air · moderate bicarbonate · Ca²?/Mg²? present
Organ/tissue perfusion
GI and cardiac prep
Platelet aggregometry
5% CO₂ incubator · high bicarbonate · Ca²? omitted
Suspension and spinner-flask culture (prevents clumping)
Suspension / spinner scale-up
Ambient air · phosphate-buffered · with or without Ca²?/Mg²?
General washing and rinsing
CMF form for dissociation
Rinsing serum before trypsinization (CMF DPBS)
Priming cells for dissociation (CMF DPBS + EDTA)
Flow cytometry / live-cell imaging buffer base (phenol-red-free DPBS)
Practical handling notes. Phenol red is not inert — it has weak estrogenic activity[1] and overlaps common fluorescence channels, so use phenol-red-free BSS for imaging, flow, and hormone-response assays. Warm to 37 °C before contact to avoid cold shock during washes. Confirm pH by indicator color or measurement before use, since bicarbonate systems drift quickly once exposed to air. For primary cells, stem cells, and gamete/embryo work, specify cell-culture-grade water, filter sterility, and a defined low endotoxin limit.
Task to Solution
Quick reference: the task decides the solution
The bench-level version of the two decisions above — read from the task you are performing.
Task
Recommended BSS
Rinse serum before trypsinization
CMF HBSS or CMF DPBS (serum inhibits trypsin, so clear it first)
Wash cells inside a CO₂ workflow
EBSS
Open-bench rinse or resuspension
HBSS
Prime cells for dissociation
CMF HBSS or CMF DPBS + EDTA
Suspension/spinner scale-up
Eagle spinner salts (Ca²?-free)
Organ perfusion, platelet work
Tyrode's
Flow cytometry / live-cell imaging buffer base
Phenol-red-free HBSS or DPBS
Drug, hormone, or dye dilution carrier
Any isotonic BSS matched to gas phase
FAQ
Frequently asked questions
The questions that come up most often when a purchasing spec meets a bench protocol.
By gas phase, not by cell line. EBSS is buffered for 5% CO₂; on the open bench it outgasses CO₂ within minutes and drifts alkaline. HBSS is buffered for ambient air; in a CO₂ incubator it over-acidifies. Match the solution to the gas phase of the work.
No. A BSS is not a growth medium. It carries no amino acids, vitamins, or serum, so it is used for washing, dissociation prep, dilution, transport, dissection, and assay setup — not for proliferation in the incubator. It holds cells and tissues at physiological pH, osmolality, and ionic balance during handling, and that is all it is designed to do.
When you are taking cells off the plate. Ca²? and Mg²? maintain cadherin (cell–cell) and integrin (cell–substrate) adhesion, so removing them primes cultures for enzymatic dissociation, usually with trypsin and EDTA. The rule of thumb: cells staying attached, keep Ca²?/Mg²?; cells being detached, remove both.
Because serum inhibits trypsin, so it has to be cleared first. Use CMF HBSS or CMF DPBS for that rinse — the same wash removes the serum and starts stripping the divalent cations that hold the junctions together.
You can, if you need CO₂-independent buffering on the bench: HEPES at 10 to 25 mM. Note that it can be phototoxic under light and cytotoxic at higher concentrations, so keep the concentration in that range and protect the solution from light.
Phenol red is not inert. It has weak estrogenic activity and overlaps common fluorescence channels, so phenol-red-free BSS is the right choice for imaging, flow cytometry, and hormone-response assays. Of the compositions on this page, Tyrode's carries no phenol red, while EBSS and HBSS carry 10 and 11 mg/L respectively.
To stop cells clumping. Spinner salts omit Ca²? specifically to prevent aggregation in suspension, which is what makes the formulation suitable for suspension and spinner-flask culture. It keeps the high bicarbonate of a 5% CO₂ formulation, so it belongs in the incubator, not on the open bench.
Key References
Supporting literature
The published sources behind the selection logic, the compositions, and the handling notes above.
Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS (1986). Phenol red in tissue culture media is a weak estrogen. PNAS 83:2496–2500.
Earle WR (1943). Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J Natl Cancer Inst 4:165–212.
Hanks JH, Wallace RE (1949). Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc Soc Exp Biol Med 71:196–200.
Waymouth C (1970). Osmolality of mammalian blood and of media for culture of mammalian cells. In Vitro 6:109–127.
Freshney RI (2015). Culture of Animal Cells, 7th ed. Wiley-Blackwell.
