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®
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?
This totally depends on the Nucleofector® system you are working with. While in the 100 µL reaction vessels usually 1 - 5 µg DNA is being used, for the 20 µL reaction vessels (e.g. X Unit, 96-well Unit and HT...
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?
We only recommend the depletion method. For Example, the RosetteSep™ Isolation Kit for human monocytes [Stem Cell Technologies, Cat. No 15028]. The advantage of depletion is that the monocytes are left untouched by antibodies during the process.
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?
The results obtained using 7AAD staining are much clearer than those obtained using propidium iodide (PI). Using flow cytrometry stained and unstained, transfected and non-transfected populations can be separated much more easily.
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®
?
The time course of plasmid expression after Nucleofection® might be different from the time course seen with other transfection methods. We recommend looking at different time points and start with the first time point as early as 4 to 6 hours post...
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®
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The PBS should be calcium and magnesium free to prevent clumping of cells. We routinely use PBS without calcium and magnesium for example Lonza Cat. No. BEBP17-516Q
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?
GFP expression has been observed for up to one week as measured by light and fluorescence microscopy.
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?
In general the overall expression level differs between different cell types. Don't expect the same level of gene induction when working with primary cells. You might need to change to other DNA amounts or ratios.
D
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s
?
Insect cells are cultivated at lower temperatures than E.coli or mammalian cells (S2 cells at 24°C, Sf9 cells at 28°C).Therefore they need a dedicated incubator with appropriate temperature.
I
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T
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N
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®
?
Yes. We recommend using mice between 6-12 weeks. Using mouse T cells isolated from younger or older animals for Nucleofection® may result in much lower transfection efficiencies and/or viabilities.
W
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s
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o
n
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n
m
a
m
m
a
l
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a
n
c
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l
l
s
?
Vectors with a CMV promoter typically work fine for transient protein expression in mammalian cells, like HEK293, CHO or NSO. For insect cells, like Sf9 or Sf21, vectors with insect promoters are required. In HEK293E (EBNA) cells, the use of...
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