NOTE: ChemoTx system microplate wells hold either 30µL or 300µL of fluid. In the following instructions volumes in parentheses refer to instruments with a well volume of 300µL.
Variations in the actual amount dispensed from pipettes, especially multi-channel pipettes, can be considerable. To help compensate for this, the ChemoTx system is designed to give good results within a range of delivered volumes; fluid in the wells can vary between 27µL and 31µL (295µL and 303µL).
To calibrate your pipette and familiarize yourself with the process of filling a ChemoTx microplate, go through the filling process using distilled water in place of experimental solutions.
- Set your pipette to dispense 29µL (299µL). The ChemoTx system is designed to allow for variations in delivered volume of ± 2µL (± 4µL) with good results.
- Pipette distilled water or experimental solutions into the wells.
- Touching only the edges of the frame, position the framed filter over the filled microplate, printed side up, with the A1 corner at the upper left. Set the frame down on the microplate so that the four pins in the microplate enter the corresponding holes in the filter frame. Press the frame down firmly at each corner until the frame makes contact with the perimeter of the microplate.
- Examine the sites to be sure the fluid makes contact with the filter at each completely filled site. Calibration figure 1 shows how each site should look from the top. There may appear to be a bubble trapped under the filter.
Calibration Figure 1, top view:
The air ring is not a trapped bubble. The seal between the rims of the wells and the under side of the filter is an aqueous seal only, not an air seal. When the cell suspension is pipetted onto the top side of the filter, one to five microliters of suspension media can flow through the filter and displace the air. (Note: the air ring may not appear as regular as shown in the illustration above.)
In Calibration Figure 2, the microplate well has been overfilled (1st diagram). Applying the filter forces excess fluid over the rims and breaks the aqueous seal (2nd diagram). When cell suspension is added to the top of the filter, the wells can leak (3rd diagram).
Calibration Figure 2, side view:
In Calibration Figure 3 the microplate well has been underfilled. The underside of the filter does not contact the fluid in the well. This prevents contact between the cell suspension above the filter and the fluid in the microplate below the filter. Air is permanently trapped in the wells. Diffusion, and therefore migration, cannot take place.
Calibration Figure 3, side view:
- We recommended an initial setting of 29µL (299µL) on your pipette, but since each pipette is different, you may need to adjust your pipette setting. Fill a column of wells and apply the filter. Look at the top surface of the filter for the dry, donut-shaped rings around the area where the filter contacts the fluid in the wells. If there are wells with no contact between the fluid in the wells and the filter, then they are underfilled. If there are wells with fluid spilling over the rims, they are overfilled. Adjust your pipette setting to compensate for the appearance of these problems.
If you are using a multichannel pipette and you have both these problems in the same column, then:
- there are leaks between one or more of the tips and the pipette;
- contamination on some of the tips is causing fluid to be dispensed unequally;
- your pipette is too inaccurate for use with the ChemoTx system; or
- several of the above.
If (a), re-seat the pipette tips; if (b), load new, clean tips; if (c) or (d), try a different pipette.If a few wells are slightly underfilled, you can gently push down the top of the filter at those sites with a clean, rounded-end glass rod (or similar instrument), until the filter makes contact with the fluid. Once contact is made, fluid from the cell suspension on top of the filter will displace air at the top of the wells.
- When the pipette is calibrated and delivering about 29µL (299µL) to each well, remove the framed filter (use a small, thin object such as a paper clip to gently loosen the frame from the corner pins), decant or aspirate the distilled water out of the wells, and use the pipette to fill the wells with the fluids to be tested in your experiment.
ChemoTx® is a registered trademark of Neuro Probe, Inc.