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Fluorescence

Chromeo™ LT Live Cell Membrane Staining Kit

long-term staining of membranes in live or fixed cells & tissues

The Chromeo™ LT Live Cell Membrane Staining Kit utilizes a proprietary, coumarin-based fluorescent complex* that rapidly diffuses into live or fixed cells and tissue. The stain accumulates in lipid structures such as plasma and internal membranes, lysosomes or lipid bodies, allowing them to be specifically labeled and imaged with low background. The use of Chromeo LT Live Cell Membrane Stain enables quick and easy staining of cellular lipid structures. Because it does not elicit toxicity, it is suitable for live cell applications. Its affinity for and retention in lipid structures and its stability towards photobleaching make it possible to monitor cells over a period of several days (see Figure 7 under the Application Data tab below).

Live NRK cells stained by Chromeo LT Live Cell Membrane Stain from Active Motif
Figure 1: Staining of live NRK cells with the Chromeo™ LT Live Cell Membrane Stain.

NRK cells were plated in 3 cm cell culture dishes and washed once with pre-warmed PBS before adding 30 nM Chromeo LT Live Cell Membrane staining solution. After incubation for 10 minutes at 37°C, cells were washed once with pre-warmed PBS and were imaged in PBS with a Nikon CLSM (60x objective).

* The Chromeo™ LT Live Cell Membrane Stain is manufactured under license by OnkoTec GmbH.

 
Name Format Cat No. Price  
Chromeo™ LT Live Cell Membrane Staining Kit 1 kit 15007 $280 Buy Now

The Chromeo™ LT Live Cell Membrane Staining Kit features a cell permeable fluorescent complex that selectively stains lipid structures like plasma and internal membranes with high specificity. It can be used to stain membranes in living cells, in paraformaldehyde fixed cells and in Formalin-Fixed Paraffin-Embedded (FFPE) tissue. The fluorescent complex shows high specificity and low background staining, enabling the use of only nanomolar concentrations for efficient staining. There is no need to pre-treat or permeabilize the cells prior to the short incubation time of just 10 minutes. After only one wash step with PBS, the stained cells are ready for image acquisition. The stain can be excited from 420-490 nm, including the 488 nm argon laser line. Because the emission range is 500-540 nm, standard FITC filter sets can be used for image capture. These fluorescent properties enable the stain to be used in combination with blue fluorescent (e.g. Hoechst) or red fluorescent (e.g. Bodipy) dyes. In addition, because the stain is non-toxic and is retained by lipids, long-term labeling experiments lasting for several days can be performed.

Chromeo LT Live Cell Membrane Stain advantages

  • Water soluble fluorescent stain for live cells, for paraformaldehyde fixed cells and for tissue
  • Low background fluorescence
  • Low toxicity
  • Easy to use
  • Excitable at 488 nm
  • Use FITC filter sets
  • Long-term labeling experiments

The Chromeo™ LT Live Cell Membrane Staining Kit contains a small fluorescent complex that readily diffuses into live or fixed cells and tissue where it stains lipid structures including the plasma membrane and internal membranes such as lysosomes, lipid bodies and the nuclear envelope. This staining pattern has been shown in a number of different cell lines such as NRK (Figure 1), HeLa (Figure 2), Caco, HTB5 and HTB9.

Live HeLa cells stained by Chromeo LT Live Cell Membrane Stain from Active Motif
Figure 2: Staining of live HeLa cells with the Chromeo LT Live Cell Membrane Stain.

HeLa cells were seeded in 3 cm cell culture dishes and washed once with pre-warmed PBS before adding 30 nM Chromeo LT Live Cell Membrane staining solution. After incubation for 10 minutes at 37°C, cells were washed once with pre-warmed PBS and immediately imaged in PBS with a Zeiss Axiovert 200 (left: 5x objective, right: 20x objective).


The Chromeo LT Live Cell Membrane Staining Kit can also be used to stain lipid structures in paraformaldehyde (PFA) fixed cells (Figure 3). Methanol fixation is not compatible with the stain as the fluorescent complex might be destroyed.

PFA-fixed  HeLa cells stained by Chromeo LT Live Cell Membrane Stain from Active Motif
Figure 3: Staining of PFA-fixed HeLa cells with the Chromeo LT Live Cell Membrane Staining Kit.

HeLa cells were seeded in µ-Chamber 12-well cell culture dishes (Ibidi GmbH, Germany). The cells were washed once with pre-warmed PBS and fixed for 15 minutes at room temperature with 3.7% PFA. After washing with PBS the cells were stained with 30 nM Chromeo LT Live Cell Membrane staining solution for 10 minutes at room temperature. The nuclei have been counterstained with DAPI (right).The cells were imaged with a Zeiss Axiovert 200 (20x objective).


The Chromeo LT Live Cell Membrane Staining Kit has been shown to stain Formalin-Fixed Paraffin-Embedded (FFPE) tissue. Tissue was treated with xylol and successively treated with 96, 80 and 70% ethanol before washing with dH2O and incubating with the lipid stain (Figure 4).

Formalin-Fixed Paraffin-Embedded (FFPE) epithelial carcinoma stained by Chromeo LT Live Cell Membrane Stain from Active Motif
Figure 4: Staining of Formalin-Fixed Paraffin-Embedded (FFPE) epithelial carcinoma with the Chromeo LT Live Cell Membrane Stain.

Fluorescence microscopy was used to study the photostability of the Chromeo LT Live Cell Membrane Stain. Again, HeLa cells were stained with 30 nM of the long-term membrane stain. The cells were continuously illuminated using the FITC filter set of the Zeiss Axiovert 200, pictures were acquired every 20 seconds. Figures 5 and 6 show the high stability of Chromeo LT Live Cell Membrane Stain, with less than a 20% decrease in fluorescence intensity after 4 minutes of permanent illumination.

Photobleaching analysis of the Chromeo LT Live Cell Membrane Stain from Active Motif
Figure 5: Photobleaching analysis of Chromeo LT Live Cell Membrane Stain.

HeLa cells were stained with 30 nM Chromeo LT Live Cell Membrane staining solution and washed once with pre-warmed PBS and illuminated continuously with a Zeiss Axiovert 200 (10x objective). Images were captured every 20 seconds; shown above are those from 20, 60, 120, 180 and 240 seconds.

Statistical analysis of the photobleaching analysis of the Chromeo LT Live Cell Membrane Stain from Active Motif
Figure 6: Statistical analysis of the photobleaching experiment shown in Figure 5.

Fluorescence intensities of the images shown in Figure 5 were quantified using the ImageJ program.


Due to its low cell-toxicity, high retention in lipid structures and high photostability, the Chromeo LT Live Cell Membrane Stain can be used in long-term labeling experiments. In the experiment shown in Figure 7, NRK cells were stained and are then imaged over a period of 72 hours.

Long-term imaging of HeLa cells stained with the Chromeo LT Live Cell Membrane Stain from Active Motif
Figure 7: Long-term imaging of HeLa cells stained with the Chromeo LT Live Cell Membrane Staining Kit.

HeLa cells were seeded in 3 cm cell culture dishes and washed once with pre-warmed PBS before adding 30 nM Chromeo LT Live Cell Membrane staining solution. After incubation for 10 minutes at 37°C, cells were washed once with pre-warmed PBS and either imaged immediately in PBS with a Zeiss Axiovert 200 (40x objective), or incubated in culture medium for an additional 24, 48 or 72 hours at 37°C before imaging.

Contents and Storage

4 vials of lyophilized fluorescent membrane stain. Each vial provides enough fluorescent complex for 30 ml of a 30 nM staining solution. Store at 4°C in the dark. Contents guaranteed 6 months when stored properly.