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HHBS (Hank's Buffer with Hepes) Preparation and Recipe

HHBS is a general purpose buffer that combines the stability of Hank's buffer with the good buffering capacity of HEPES buffering solution (one of Good's buffers). HHBS is used extensively for cell culture, but is sensitive to light.

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To prepare  L of HHBS (Hank's Buffer with Hepes):
Change the value in the textbox above to scale the recipe volume

Table 1. Required components
Calcium Chloride (mw: 110.98 g/mol)0.14 g0.00126 M
Magnesium Chloride Hexahydrate (mw: 203.30 g/mol)0.1 g0.00049 M
Magesium Sulfate Heptahydrate (mw: 246.47 g/mol)0.1 g0.00041 M
Potassium Chloride (mw: 74.55 g/mol)0.4 g0.00533 M
Potassium Phosphate Monobasic (mw: 136.09 g/mol)0.06 g0.00044 M
Sodium bicarbonate (mw: 84.01 g/mol)0.35 g0.00417 M
Sodium chloride (mw: 58.44 g/mol)8 g0.138 M
Sodium Phosphate Dibasic (mw: 141.96 g/mol)0.048 g0.00034 M
D-Glucose (Dextrose) (mw: 180.16 g/mol)1 g0.00556 M
Hepes (mw: 238.30 g/mol)4.76 g0.02 M
  1. Prepare 800 mL of distilled water in a suitable container.
  2. Add 0.14 g of Calcium Chloride to the solution.
  3. Add 0.1 g of Magnesium Chloride Hexahydrate to the solution.
  4. Add 0.1 g of Magesium Sulfate Heptahydrate to the solution.
  5. Add 0.4 g of Potassium Chloride to the solution.
  6. Add 0.06 g of Potassium Phosphate Monobasic to the solution.
  7. Add 0.35 g of Sodium bicarbonate to the solution.
  8. Add 8 g of Sodium chloride to the solution.
  9. Add 0.048 g of Sodium Phosphate Dibasic to the solution.
  10. Add 1 g of D-Glucose (Dextrose) to the solution.
  11. Add 4.76 g of Hepes to the solution.
  12. Add distilled water until the volume is 1 L.

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Physiological Buffer
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Sample Preparation
Cell/Culture/Growth Media
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This online tool may be cited as follows


"Quest Calculate™ HHBS (Hank's Buffer with Hepes) Preparation and Recipe." AAT Bioquest, Inc.28 May2024https://www.aatbio.com/resources/buffer-preparations-and-recipes/hhbs-hanks-buffer-with-hepes.


AAT Bioquest, Inc. (2024May 28). Quest Calculate™ HHBS (Hank's Buffer with Hepes) Preparation and Recipe. AAT Bioquest. https://www.aatbio.com/resources/buffer-preparations-and-recipes/hhbs-hanks-buffer-with-hepes.

This online tool has been cited in 1 publications, including

Identification of Autophagy as a Functional Target Suitable for the Pharmacological Treatment of Mitochondrial Membrane Protein-Associated Neurodegeneration (MPAN) In Vitro
Journal: Pharmaceutics (2023): 267