How to make phosphate-buffered saline (PBS)
Standard 1× and 10× PBS recipes with exact weigh-outs, the final ion concentrations, and the reason 10× stock precipitates in the cold.
1× PBS 1 L of 1× PBS
Preparation time about 15 minutes.
| Component | Formula weight | Amount |
|---|---|---|
| NaCl | 58.44 g/mol | 8.00 g |
| KCl | 74.55 g/mol | 0.20 g |
| Na₂HPO₄ (anhydrous) | 141.96 g/mol | 1.44 g |
| KH₂PO₄ | 136.09 g/mol | 0.24 g |
| Deionised water | — | to 1 L |
Procedure
- Dissolve the four salts in about 800 mL of deionised water, adding them one at a time and letting each dissolve fully.
- Check the pH. It should land within about 0.1 unit of 7.4 with no adjustment. If it is off, adjust with HCl or NaOH — but first suspect a weighing error, because the recipe is self-buffering.
- Make up to 1 L.
- Autoclave, or filter through 0.22 µm for cell culture.
What matters here
10× stock precipitates at 4 °C
Phosphate salts are much less soluble cold. A 10× stock left in the fridge will throw crystals. Warm it to room temperature and stir until clear before diluting; if you dilute while crystals are present, your 1× will be short on phosphate.
pH shifts on dilution
10× PBS is not pH 7.4 — it sits nearer 6.8 because of ionic-strength effects. That is normal. The pH corrects itself when you dilute to 1×.
With or without Ca²⁺ and Mg²⁺
The recipe above is the calcium- and magnesium-free version, which is what you want for washing cells before trypsinisation — divalent cations promote adhesion. Add 0.1 g/L CaCl₂ and 0.1 g/L MgCl₂·6H₂O for the complete version.
Frequently asked questions
Cloudy after autoclaving
Usually calcium phosphate precipitation if you used the complete formulation. Autoclave the divalent cations separately and add them after cooling.
Cells detaching during washes
You are probably using PBS with Ca²⁺/Mg²⁺ when you wanted the plain version, or the PBS is too cold.