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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).

What is the lowest amount of Human Hepatocytes that can be used with the 96-well Shuttle™ System or the 4D-Nucleofector™ X-Unit?

Human hepatocytes do not like to be cultured and transfected at low densities. Therefore, using less than 100,000 cells per Nucleofection™ is not recommended.

How long does it take to obtain stable transfectants?

Depending on individual cell type and doubling rate, selection of stable transfectants will take between 7 and 28 days. Expansion and characterization of single cell clones will take several weeks in addition.

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.

I have a very high transfection efficiency but most of my cells die during selection, is this to be expected?

This is the normal pattern you should expect to see. Only a small proportion (1/10,000 to 1/100) of all transfected cells will integrate the transfected DNA into their genome and become stable transfectants. The remaining cells lose the transfected...

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).

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...

What kind of effects could a mycoplasma contamination have on my cells?

"Mycoplasma contamination can: > Inhibit cell metabolism > Cause cell death > Induce morphological alterations > Alter DNA, RNA, and...

Lonza recommends using PEG purified DNA for Nucleofection™ of DC's and macrophages. Is it necessary to perform the PEG purification step for the positive control plasmid pmaxGFP™ supplied in each Nucleofector™ Kit?

No, it is not. The PEG purification step removes additional endotoxin from the DNA preparation. The pmaxGFP reporter plasmid supplied in each kit is highly purified and endotoxin levels are <0.01E.U./µg DNA (which means <1pg endotoxin/µg DNA).

How can I detect mycoplasma contamination of my cells?

We recommend using the 20-minute MycoAlert™ Mycoplasma Detection Kit from Lonza. The simple four-step MycoAlert™ Assay is a selective biochemical test that exploits the activity of mycoplasmal enzymes. The presence of these enzymes provides a rapid...

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