Monday, January 4, 2010

Automated Cell Dispensing into 1536-Well Microplates for HTS Screening

Today’s HTS demands have moved screening assays towards high well density plates as well as placed more emphasis on cell based assays in lieu of the conventional biochemical determinations. High density plates allow more samples to be assayed simultaneously, conserve reagents and lower assay costs. The use of 1536-well microplates for cell based assays requires the use of accurate and reliable automation in order to dispense uniform numbers of cells to each microplate well in a volume of a few microliters. We have used the MicroFlo Select Peristaltic pump dispenser to dispense cells into 1536-well microplates. As with any cell based experiment, providing uniform numbers of viable cells is paramount to successful experiments. CHO-M1 cells (200 cells / µL) were dispensed into 1536-well microplates (4 µL/well) and cell uniformity as measured by the luminescent determination of ATP using CellTiter-Glo assay kits from Promega Corporation. Luminescent signal for wells not containing cells averaged 10, while those wells with 800 cells averaged 23210 with a Z’ =0.753 (Table 1).

Whole plate Dispense-statistics.

Table 1. Whole plate Dispense-statistics. The mean, standard deviation and Z’ value for Cell Titer Glo data generated from two 1536-well plates. One plate received 4 µL of media only while the second received 4 µL of cell suspension containing 200 cells/µL.

As demonstrated in Figure 1, the signal was uniform across the entire plate. The addition of different volumes of a cell solution demonstrates the dispense accuracy and linearity of MicroFlo Select.


Uniformity of Dispensing into 1536-well Microplates


Figure 1. Uniformity of Dispensing into 1536-well Microplates. Surface plot of the data generated from a Cell Titer Glo luminescent assay. A MicroFlo Select was used to dispense 4 µL of CHO-M1 cell suspension followed by 4 µL of CellTiter Glo reagent to all the wells of a 1536-well microplate.

As seen in Figure 2, the relationship between cell numbers dispensed (as determined by the fluid volume) correlates very well with the signal generated from a CellTiter-Glo assay. These data also show that volumes as low as 1 µL can be reliably programmed.


Linearity of Dispense


Figure 2. Linearity of Dispense. The MicroFlo Select was used to dispense different volumes of a cell suspension (200 cells/ µL) into 1536-well microplates followed by the addition of media to a final volume of 4 µL. Subsequent to the cell dispense, 4 µL of CellTiter Glo reagent was added using the MicroFlo Select and the luminescence was determined. Linear regression analysis was then performed on the data.

In addition to being accurately dispensed, these cells remain sterile and viable. Pictures taken the day following dispense indicate that the cells are viable as well as uniformly dispersed (Figure 3). The MicroFlo’s autoclavable tubing cassette allows the entire fluid path to be easily sterilized.

Statistical Comparison of the Luminescent Signal with Different Cell Numbers

Table 2. Statistical Comparison of the Luminescent Signal with Different Cell Numbers

Representative images of H-mesotheoma cells dispensed into 1536-well plates using a MicroFlo Select

Figure 3. Representative images of H-mesotheoma cells dispensed into 1536-well plates using a MicroFlo Select. Digital light transmission images were taken with a Zeiss inverted microscope configured with a Nikon camera.

Not everyone uses 1536-well microplates, as it is primarily the tool of the HTS laboratories only. However those that do use this plate format require accuracy and precision at low dispense-volumes. The MicroFlo Select meets these requirements, but is also capable of dispensing fluid to 96- and 384-well microplates without any hardware modification.

What are your needs for automated dispensing of cells and/or other solutions? Do you currently work in 96 or 384 formats? Any plans to move to higher well densities?

By, BioTek Instruments

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