Guidelines for Selecting Balanced Salt Solutions

Balanced Salt Solutions (BSS) are core reagents in cell biology research, widely used for cell washing, tissue dissociation, reagent dilution, and short-term cell maintenance. They mimic in vivo physiological conditions to stabilize the extracellular environment for isolated cells or tissues.

This guideline introduces mainstream BSS formulations (PBS, DPBS, HBSS, EBSS), provides component comparison tables, and offers selection strategies based on component functions and application scenarios. It also includes storage and usage precautions to help researchers select appropriate BSS efficiently.

Table of Contents

1. What Is a Balanced Saline Solution?

2. Introduction to Mainstream BSS

3. Formulation Comparison

4. Selection Guidelines for BSS

5. BSS Handling and Usage Precautions

01 What Is a Balanced Saline Solution?

Balanced Salt Solution (BSS) is a class of isotonic aqueous solutions formulated with inorganic salts, and it serves three core functions:

(1) Osmotic pressure maintenance: Prevents cell swelling or shrinkage induced by osmotic stress.

(2) pH stabilization: Provides a physiological weakly alkaline environment (pH 7.2–7.6) suitable for cell survival.

(3) Essential ion supply: Delivers necessary inorganic cations including Na⁺, K⁺, Ca²⁺, and Mg²⁺.

Note: BSS contains no nutrients and can only maintain cell viability for a short period; they must never be used as a substitute for culture medium in long-term cell culture.

02 Introduction to Mainstream BSS

2.1 PBS and DPBS

In the late 19th century, physiologists studying the maintenance of cell viability in vitro found that 0.9% Normal Saline (Cat. No.: PB180353) could only sustain osmotic pressure equilibrium but lacked the buffering capacity to counteract acidic metabolic byproducts. Under such conditions, rapid pH fluctuations frequently led to quick cell death, prompting the development of pH-stabilizing buffer systems. Between the 1930s and 1940s, as tissue culture techniques advanced, the core formulation of Phosphate-Buffered Saline (PBS) was finalized: NaCl is included to maintain isotonicity, a buffer pair consisting of NaH₂PO₄ and KH₂PO₄ stabilizes pH, and KCl is added to supply essential cations.

In the 1950s, Italian virologist Renato Dulbecco identified that non-standardized PBS formulations exhibited substantial batch-to-batch variability, compromising experimental reproducibility. To address this issue, he introduced two key modifications: first, he precisely standardized phosphate concentrations to improve buffer stability; second, he systematically defined optional supplementary components, including Ca²⁺ and Mg²⁺, glucose, sodium pyruvate, and phenol red indicator, allowing the base formulation to be adapted into multiple variants tailored to diverse experimental needs. This standardized formulation was subsequently named Dulbecco's PBS (DPBS).

To date, DPBS has evolved into a systematic product portfolio covering multiple formulation variants, including those with or without Ca²⁺ and Mg²⁺, with or without glucose, with or without sodium pyruvate, and with or without phenol red.

By contrast, standard PBS is generally only available in basic formulations, with relatively limited functional applications. Both PBS and DPBS are widely used for routine procedures, including cell washing, antibody dilution, immunoassays, protein extraction, and as a dissolution medium for reagent preparation.

2.2 HBSS and EBSS

HBSS and EBSS were developed nearly contemporaneously with PBS. In the 1940s, physiologist John H. Hanks observed that PBS lacked sufficient pH stability under the acidic conditions generated by cellular metabolism, and could not maintain effective buffering equilibrium in CO₂ culture environments. To address this limitation, he modified the buffering system: he introduced NaHCO₃ as the core buffering component and added glucose as a short-term energy source, and ultimately developed Hanks' Balanced Salt Solution (HBSS), which markedly improved pH stability during cell culture procedures.

At the same time, Wilbur Earle developed an alternative balanced salt solution system. He adjusted the Na⁺- K⁺ ratio and incorporated a higher concentration of NaHCO₃ to optimize buffering capacity for compatibility with 5%–10% CO₂ culture environments. This formulation was designated Earle’s Balanced Salt Solution (EBSS).

The core formulations of HBSS and EBSS have been used for nearly a century, both retaining the characteristic "NaHCO₃ buffer + glucose energy source" configuration, with optional supplementation of components including Ca²⁺ and Mg²⁺ and phenol red. EBSS contains a higher NaHCO₃ content and stronger buffering capacity, making it suitable for extended incubation in CO₂ incubators. HBSS has a slightly lower NaHCO₃ content and more gradual pH drift characteristics, rendering it suitable for short-term cell washing and tissue transfer operations in open environments.

03 Formulation Comparison

3.1 Formulation Comparison Between PBS and DPBS

Table 1. Formulation Comparison Between PBS and DPBS

3.2 Formulation Comparison Between HBSS and EBSS 

Table 2. Formulation Comparison Between HBSS and EBSS

04 Selection Guidelines for BSS

4.1 Selection by Component-Function: Tailored to Experimental Requirements

Table 3. Recommendations BSS by Composition

4.2 Selection by Application Scenario: Precisely Matched to Experimental Purpose

Table 4. Recommended BSS by Application Scenario

05 BSS Handling and Usage Precautions

(1) For cell and tissue washing, Ca²⁺&Mg²⁺-free, phenol red-free D-PBS (Cat. No.: PB180329) and PBS (Cat. No.: PB180327) are the most widely options;

(2) For fluorescence or colorimetric assays, Ca²⁺ & Mg²⁺-free, phenol red-free are mandatory to avoid background signal interference;

(3) For cell incubation procedures performed in a 5%–10% CO₂ incubator, EBSS is the recommended.

(4) All ready-to-use liquid BSS sold by Procell® are pre-sterilized. Opened solutions should be used as soon as possible, and aseptic technique must be observed to prevent microbial contamination. For powdered BSS products, sterile filtration through a 0.22 μm filter membrane is required immediately after reconstitution and before use.

(5) HBSS and EBSS contain NaHCO₃ as a buffering component. Once opened, these solutions should be used promptly to prevent CO₂ outgassing, which would lead to pH fluctuations.

(6) Formulations in this document are based on Procell’s products. Formulations of similar products from other manufacturers may vary; always refer to the respective manufacturer's official documentation when using other brands.