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Following Nucleofection™, I cannot obtain stable clones from single cells, what could be the reason?

Some cell lines need certain cell densities to proliferate. For those cells you can try using conditioned medium for setting up limiting dilution or culturing single cells together with feeder cells.

What are mycoplasmas?

Mycoplasmas belong to the family Mollicutes, which includes Acholeplasma, Ureaplasma and other species. However the term "Mycoplasma" is most often used as a "cover-all". More than 180 species have been identified of which 20 distinct Mycoplasma and...

What antibiotic concentration should I use for selecting stable transfectants?

Please click on the associated file.

How much DNA should I use in my Nucleofection™ Reaction to obtain stable clones?

You should use the amount of DNA recommended in the optimized protocol for transient transfection. If obtaining a very high number of clones is important for your experiments, you can try increasing the amount of DNA. For further tips see our...

How often do mycoplasma contaminations occur in cell cultures?

A conservative estimate states that between 15-35% of all continuous cell cultures are contaminated with mycoplasma (Drexler HG, Uphof CC; 2002), some estimates are even higher (up to 80 % in some countries (Koshimizu K, Kotani H; 1981).

What are useful methods to enrich/purify specific hematopoetic cell populations before Nucleofection™?

Specific blood cell populations can be enriched/purified for Nucleofection™ by MACS™ selection Kits (Miltenyi Biotec GmbH), RosetteSep™ Kits (StemCell Technologies Inc.) or Dynal™ beads (Invitrogen Corporation).

Is there an advantage of the MACS™ Selection Kit (Miltenyi Biotec GmbH) compared to the RosetteSep™ Kit (StemCell Technologies Inc)?

The RosetteSep Kit is less expensive than the MACS Selection Kit. The MACS Selection Kit results in a higher purification (up to 97-98%) of the specific cell population.

Do you recommend a specific purification method for the selected hematopoetic cell population before Nucleofection™?

For obtaining highly purified cell populations, we always recommend using the MACS™ Selection Kit (Miltenyi Biotec GMbH).

Which luminometer is suited for performing the MycoAlert™ Assay?

With the Lucetta™ Luminometer Lonza offers a perfect match, because this small, portable single tube luminometer is designed with a tailor-made mode for running the MycoAlert™ Assay. Please find attached our list with luminometers we know to be...

MACS™ Selection Kits (Miltenyi Biotec GmbH) offer the opportunity to purify specific blood/immune cell populations by positive selection via antibody-binding or by negative selection using depletion. Which MACS purification method is preferred by Lonza?

We recommend using negative selection (depletion) because your purified cells are â??untouchedâ?, not influenced, and strictly not activated. Positive selection may cause increasing amounts of dead cells and could also lead to activation of the...

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