Physiological Buffer preparation

Tyrode’s Solution, Acidic Preparation

To prepare 1L of Tyrode’s Solution, Acidic, the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	8 g	0.1368 M
KCl (mw: 74.55 g/mol)	200 mg	0.0027 M
CaCl2•2H2O (mw: 147.01 g/mol)	240 mg	0.0016 M
MgCl2•6H2O (mw: 203.3 g/mol)	100 mg	0.0005 M
Glucose (mw: 180.16 g/mol)	1 g	0.0056 M
Polyvinylpyrrolidone (PVP) (mw: 40000 g/mol)	4 g	0.0001 M

Tyrode’s Solution, Acidic Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8 g of NaCl to the solution.
3. Add 200 mg of KCl to the solution.
4. Add 240 mg of CaCl2•2H2O to the solution.
5. Add 100 mg of MgCl2•6H2O to the solution.
6. Add 1 g of Glucose to the solution.
7. Add 4 g of Polyvinylpyrrolidone (PVP) to the solution.
8. Add distilled water until volume is 1 L.
9. Prepare at room temperature and adjust to pH 2.5 with Analar HCl (BDH). The PVP is added to increase viscosity and reduce embryo stickiness. Filter-sterilize and store in aliquots at -20?C. Acidic Tyrode?s is also available from Sigma and Specialty Media.

PBS with Ca2+and Mg2+ (D-PBS) Preparation

To prepare 1L of PBS with Ca2+and Mg2+ (D-PBS), the following components are required;

Component	Amount	Concentration
CaCl2 (mw: 110.98 g/mol)	100 mg	0.0009 M
KCl (mw: 74.55 g/mol)	200 mg	0.0027 M
MgCl2 · 6H2O (mw: 203.3 g/mol)	100 mg	0.0005 M
NaCl (mw: 58.44 g/mol)	8 g	0.1368 M
Na2HPO4 (mw: 141.96 g/mol)	2.16 g	0.0152 M

PBS with Ca2+and Mg2+ (D-PBS) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 100 mg of CaCl2 to the solution.
3. Add 200 mg of KCl to the solution.
4. Add 100 mg of MgCl2 · 6H2O to the solution.
5. Add 8 g of NaCl to the solution.
6. Add 2.16 g of Na2HPO4 to the solution.
7. Add distilled water until volume is 1 L.
8. Filter-sterilize. Store at 25?C for up to 12 mo.

PBS (Phosphate Buffered Saline) (1X, pH 7.4) Preparation

PBS is an isotonic buffer frequently used in biological applications, such as washing cells, transportation of tissues, and dilutions. PBS closely mimics the pH, osmolarity, and ion concentrations of the human body. Since it is nontoxic to cells, it is extensively used for cell container rinsing and other preparations that might leave a residue. It is simple to prepare and has good shelf life, but will precipitate in the presence of zinc ions.

To prepare 1L of PBS (Phosphate Buffered Saline) (1X, pH 7.4), the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.4 g/mol)	8 g	0.137 M
KCl (mw: 74.551 g/mol)	200 mg	0.0027 M
Na2HPO4 (mw: 141.96 g/mol)	1.44 g	0.01 M
KH2PO4 (mw: 136.086 g/mol)	245 mg	0.0018 M

PBS (Phosphate Buffered Saline) (1X, pH 7.4) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8 g of NaCl to the solution.
3. Add 200 mg of KCl to the solution.
4. Add 1.44 g of Na₂HPO₄ to the solution.
5. Add 245 mg of KH₂PO₄ to the solution.
6. Adjust solution to desired pH (typically pH ≈ 7.4).
7. Add distilled water until volume is 1 L.

HHBS (Hank’s Buffer with Hepes) Preparation

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. To purchase, click here.

To prepare 1L of HHBS (Hank’s Buffer with Hepes), the following components are required;

Component	Amount	Concentration
CaCl2 (mw: 110.98 g/mol)	140 mg	0.0013 M
MgCl2-6H2O (mw: 203.3 g/mol)	100 mg	0.0005 M
MgSO4-7H2O (mw: 246.47 g/mol)	100 mg	0.0004 M
KCl (mw: 74.55 g/mol)	400 mg	0.0053 M
KH2PO4 (mw: 136.086 g/mol)	60 mg	0.0004 M
NaHCO3 (mw: 84.01 g/mol)	350 mg	0.0042 M
NaCl (mw: 58.44 g/mol)	8 g	0.138 M
Na2HPO4 (mw: 141.96 g/mol)	48 mg	0.0003 M
D-Glucose (Dextrose) (mw: 180.156 g/mol)	1 g	0.0056 M
Hepes (mw: 238.3 g/mol)	4.76 g	0.02 M

HHBS (Hank’s Buffer with Hepes) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 140 mg of CaCl₂ to the solution.
3. Add 100 mg of MgCl₂-6H₂O to the solution.
4. Add 100 mg of MgSO₄-7H₂O to the solution.
5. Add 400 mg of KCl to the solution.
6. Add 60 mg of KH₂PO₄ to the solution.
7. Add 350 mg of NaHCO₃ to the solution.
8. Add 8 g of NaCl to the solution.
9. Add 48 mg of Na₂HPO₄ 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 volume is 1 L.

HEPES Buffer (1 M, 7.5 pH) Preparation

HEPES buffer is one of the Good’s zwitterionic buffer with a pH range of 6.8-8.2. It is commonly used in cell culture medium as its pH is well maintained with changes in carbon dioxide concentration.

To prepare 1L of HEPES Buffer (1 M, 7.5 pH), the following components are required;

Component	Amount	Concentration
HEPES (mw: 238.30 g/mol)	238.3 g	1 M

HEPES Buffer (1 M, 7.5 pH) Preparation steps

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 238.3 g of HEPES to the solution.
3. Adjust solution to desired pH by 10N NaOH.
4. Add dH2O until volume is 1 L.

HBSS (Hank’s Balanced Salt Solution) Preparation

Maintains physiological pH and osmotic balance for cells. To prepare 1L of HBSS (Hank’s Balanced Salt Solution), the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	8 g	0.14 M
KCl (mw: 74.55 g/mol)	400 mg	0.005 M
CaCl2 (mw: 110.98 g/mol)	140 mg	0.001 M
MgSO4-7H2O (mw: 246.47 g/mol)	100 mg	0.0004 M
MgCl2-6H2O (mw: 203.303 g/mol)	100 mg	0.0005 M
Na2HPO4-2H2O (mw: 177.99 g/mol)	60 mg	0.0003 M
KH2PO4 (mw: 136.086 g/mol)	60 mg	0.0004 M
D-Glucose (Dextrose) (mw: 180.156 g/mol)	1 g	0.006 M
NaHCO3 (mw: 84.01 g/mol)	350 mg	0.004 M

HBSS (Hank’s Balanced Salt Solution) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8 g of NaCl to the solution.
3. Add 400 mg of KCl to the solution.
4. Add 140 mg of CaCl₂ to the solution.
5. Add 100 mg of MgSO₄-7H₂O to the solution.
6. Add 100 mg of MgCl₂-6H₂O to the solution.
7. Add 60 mg of Na₂HPO₄-2H₂O to the solution.
8. Add 60 mg of KH₂PO₄ to the solution.
9. Add 1 g of D-Glucose (Dextrose) to the solution.
10. Add 350 mg of NaHCO₃ to the solution.
11. Add distilled water until volume is 1 L.

Extracellular Saline (1x, pH 7.3) Preparation

To prepare 1L of Extracellular Saline (1x, pH 7.3), the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	8.182 g	0.14 M
KCl (mw: 74.55 g/mol)	402 mg	0.0054 M
CaCl2 (mw: 110.98 g/mol)	294 mg	0.002 M
MgSO4 (mw: 120.37 g/mol)	1.204 g	0.01 M
HEPES (mw: 238.3 g/mol)	1.191 g	0.005 M
Glucose (mw: 180.16 g/mol)	1.802 g	0.01 M

Extracellular Saline (1x, pH 7.3) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8.182 g of NaCl to the solution.
3. Add 402 mg of KCl to the solution.
4. Add 294 mg of CaCl2 to the solution.
5. Add 1.204 g of MgSO4 to the solution.
6. Add 1.191 g of HEPES to the solution.
7. Add 1.802 g of Glucose to the solution.
8. Add distilled water until volume is 1 L.