heliXcyto
Troubleshooting
The normalization peak should be a sharp injection and ejection of Normalization solution, with a plateau ideally in the same fluorescent count range as the highest measurement data. If the normalization peak injection or ejection is not precise the tube passivation could be uneven, air bubbles might interrupt the fluorescent solution, a pump could have stopped working, or the normalization solution could be too old. First, prepare a fresh normalization solution in RB1. Make sure you have added detergent to the running buffer. We recommend 0.01 % Pluronic, which is suitable for living cells. If that does not solve the issue, restart the device.
New heliXcyto chips have a raw fluorescence below 3 x 105 cps in the measurement channel when performing a chip test (LED = 0.5). Stable adherence of fluorescently labeled analytes to the chip material (glass, gold, polymer) might lead to an increase of this background fluorescence which can be noted by a difference of raw fluorescence counts before and after the measurement, as well as increasing signals in repeated chip tests. As the single photon counters are damaged by too high fluorescence, the shutter might close for protective reasons if the analyte signal on top of a high chip background reaches more than 2 x 106 cps. In this case you can lower the LED power in your next run, or reduce the analyte concentration. Make sure to clean the chip before storage with the Chip cleaning kit to prevent excessive fluorophore build-up.
There are several ways to avoid a drift of the signal. First, lower the LED excitation power to below 0.5. If the drift is already visible in the baseline, you can perform a Cyto chip test which includes a bleaching step. If the signal still drifts, you should thoroughly clean the device by performing 1-3 x System Washes. Do not use the camera during a measurement.
Consider the measurement time in relation to the koff rate. Maybe you are working with a very strong binder and need to extend the dissociation phase. A technical reason might be the capture of autofluorescent dirt particles (often visible in the snapshots). Make sure all bottles, vials and microtiter plates are free from dust (if necessary, rinse them with water before use) and filter or centrifuge all buffers and solutions before use. If a malfunction of the buffer pump is the reason (no washout of analyte, which is visible in the raw data on both spots), please try to restart the device.
Ensure that your cell suspension is monodisperse (no clumping) and has a concentration of 1 x 106 cells/mL or higher. Resuspend the cell suspension freshly before inserting the samples into the heliXcyto biosensor. You can filter the cell suspension through a cell strainer to reduce clumping. Especially when working with small cells, increasing the cell concentration up to 10 x 106 cells/mL may enhance the capture efficiency.
Thoroughly wash the cell suspension before the measurement and resuspend it by pipetting up and down or pass it through a strainer/filter. In order to avoid aggregation, you can also dilute the cell suspension. Use buffer without Ca2+ and Mg2+ such as our RB1 and/or add EDTA.
Restart the device. If this does not help, contact our customer support. As a general note, you should always insert the sample tray and the chip tray completely, i.e. do not instantly stop once you encounter a slight resistance