Cell Proliferation Reagent WST-1 Protocol & Troubleshooting

Product No. CELLPRO-RO

Protocol

Settlement of cells

Cells are not required to settle down to the bottom of the well as cells do not interfere with the measurement. Cells do not absorb, at the wavelength used for quantification of WST-1 cleavage. It is therefore possible to measure immediately after shaking without any further step, as described in the protocol in the package insert.

Use with yeast or bacteria

There is no data for using these kits for the measurement of bacterial or yeast proliferation; the kits have also not been developed for this purpose. The easiest way to measure bacterial or yeast proliferation would be to determine the OD.

Simultaneous use of Cell Proliferation Reagent WST-1 and Cell Proliferation ELISA BrdU, colorimetric

Figure 52a of the Roche ′Apoptosis, Cytotoxicity and Cell Proliferation Manual′, 4th edition shows the combined use of the Cell Proliferation Reagent WST-1 and the Cell Proliferation ELISA BrdU, colorimetric for simultaneous measurement of cell viability and cell proliferation. A protocol is given in the figure legend. To perform assays simultaneously one must order both kits, additional material is listed in the pack inserts.

Simultaneous use of Cell Proliferation Reagent WST-1, Cell Proliferation Kit I (MTT) and Cell Proliferation Kit II (XTT)

The MTT/XTT/WST-1 assays utilize different tetrazolium salts to measure metabolic activity in active cells. Each kit requires a different wavelength to detect metabolic activity, MTT 550 nm, XTT 492 nm, and WST-1 420-480 nm. Discrimination of the signal from each salt would be difficult. Sensitivity and linear detection range of established kits run independently would be compromised with use of combined salts MTT, XTT, and WST-1. Therefore, simultaneous use of MTT, XTT, and WST-1 is not recommended.

Stopping of the WST-1 reaction

Stopping the reaction of the Cell Proliferation Reagent WST-1 is possible by either freezing the sample or by adding 1% Triton X-100/ 0.1% SDS (final concentration) for 5 min at room temperature (+15 to +25 °C or +37 °C).

Troubleshooting

Potential interference of Phenol Red and/or FCS

Phenol Red will increase the absorbance by no more than 0.1 OD units; however, the negative controls make it possible to compensate for this. The addition of up to 10% FCS also causes no problems. Roche has not systematically tested the kit for a tolerance range for Phenol Red or FCS. Higher amounts of FCS should also cause no problems. In general, the negative control should always be treated in the same way as the samples.

Materials

     
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