Nucleus-RFP

CellLight^TM Nucleus-RFP

Invitrogen (Thermo-Fisher) C10603

1 mL - received 10/26/23 - purchased for Bioimaging

Store in refrigerator

manual

Simplified protocol:

Starting concentration = 10⁸ particles per mL

Working concentration = 10 - 50 particles per cell

Use low-passage number cells;

estimate number of cells at time of treatment - should be no more than 70% confluent

(number of cells x ~30 particles/cell)/10⁸ particles/mL = mL CellLight to use

Mix thoroughly but gently with cell medium

Image cells after about 16 hours

To stain	cells
at	particles per cell (between 10 & 50)
add	µL Nucleus-RFP

From the mfgr:

CellLight Nucleus-RFP, BacMam 2.0, provides an easy way to label nuclei with red fluorescent protein (RFP) in live cells. Simply add the reagent to your cells, incubate overnight, and the cells are ready to image in the morning.

This ready-to-use construct is transfected into cells using BacMam 2.0 technology, where it expresses RFP fused to the SV40 nuclear localization sequence. You can observe nucleus-RFP behavior in live cells without the cellular toxicity associated with intercalators and label with multiple tracking or tracing dyes to image dynamic cellular processes.

Cells expressing CellLight constructs can also be fixed with formaldehyde for multiplexed imaging using immunocytochemical techniques.

CellLight Technology is:

• Fast and convenient: simply add CellLight reagent to your cells, incubate overnight, and image—or store frozen, assay-ready cells for later use
• Highly efficient: up to 90% transduction of a wide range of mammalian cell lines, including primary cells, stem cells, and neurons
• Flexible: co-transduce more than one BacMam reagent for multiplex experiments or co-localization studies; tightly control expression levels by simply varying the dose
• Less toxic: CellLight reagents are non-replicating in mammalian cells and are suitable for biosafety level (BSL) 1 handling

BacMam Technology

CellLight Nucleus-RFP, BacMam 2.0, is a fusion construct of SV40 nuclear localization sequence and TagRFP, providing accurate and specific targeting to cellular nucleus-RFP. This fusion construct is packaged in the insect virus baculovirus, which does not replicate in human cells and is designated as safe to use with biosafety level (BSL) 1 in most laboratories. BacMam technology ensures that most mammalian cell types are transduced/transfected with high efficiency and minimal toxicity. This transient transfection can be detected after overnight incubation for up to five days—enough time to carry out most dynamic cellular analyses. Like any transfection/transduction technique, the BacMam method does not transfect/transduce all of the cells with equal efficiency, making it poorly suited to cellular population studies or automated imaging/counting. CellLight reagents are ideal for experiments where cellular or subcellular co-locatization is required, or for cellular function studies that need special resolution.

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