Best Pipetting Practices for Incucyte® Lentivirus Titer Assay

The reproducibility of cell-based assays can be improved by paying close attention to practical details during sample preparation. Adjusting your pipetting practices as is required by each step of the process makes a difference (i.e., removing cell culture media with a lower aspiration speed avoids cell detachment from vessels).

In this application note, we use the protocol for a lentivirus titer assay as an example of the importance of correct pipetting techniques for each step. Learn the influence of sample temperature for pipetting results as well as recommendations for pipetting small volumes.

Frequently asked questions

  • How are bubbles prevented in cell culture?
    • Using the reverse pipetting technique - or multi-dispensing mode - helps avoid the creation of bubbles. Bubbles are often created during blow-out which is necessary for accurate pipetting when using the forward pipetting technique. Reverse pipetting and multi-dispensing modes aspirate excess volume which is left inside the tip after dispensing so no bubbles are created.
  • How can adherent cell detachment be avoided while changing culture media?
    • Slower liquid flow helps. When using an electronic pipette to remove culture media, use a slower speed setting and choose a pipette with smaller nominal (maximum) volume.
  • How to reduce shear stress to cells during pipetting?
    • Use slow liquid flow speeds and wide bore pipette tips.
  • How much extra volume should be prepared when dispensing reagent from a reagent vessel or reservoir using multi-dispensing mode with a multichannel electronic pipette?
    • Prepare 30% of excess volume to avoid aspirating air into the pipette tips of the multichannel pipette. Make sure tips are under the liquid surface before aspirating. For 10 or 120 µL multichannel pipettes, having the tips 1-3 mm below liquid surface is sufficient. For larger volumes such as 1,200 µL multichannel pipettes, the tips should be 3-5 mm below the surface. The depth is dependent on total reservoir size.
  • How to speed up sample preparation of cell-based assays?
    • Creating a master plate speeds up the addition of different reagents (or different concentrations of a reagent) onto a 96 or 384 well plate, whereas a reagent reservoir can only be used to dispense one type of solution. A master plate of reagents is prepared on a multiwell plate for easy dispensing using a multichannel pipette onto the assay plate. A master plate in a 96 well format should include at least 10% excess in each well.

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