The plasma membrane structure is a lipid bilayer that separates the interior of the cell from the exterior of the cell. Here we describe cell membrane markers that outline the cell boundary for fluorescence imaging. Fluorescent labeled lectins (e.g., wheat germ agglutinin (WGA)) can identify cell types or cellular components in live and fixed cells. Fluorescent fusion proteins can be used to follow cellular dynamics in live cells or for end-point analysis. Some plasma membrane stains can also be used as a segmentation tool for HCS assays.
Plasma membrane introduction
The plasma membrane, also known as the cell membrane, separates the interior of the cell from the extracellular environment. It is composed of about 50% lipids and 50% proteins. Cell membrane lipids, consisting of phospholipids but also glycolipids and sterols, are primarily responsible for the structural integrity of the cell membrane and controlling the movement of substances—ions and organic molecules—in and out of the cell [1]. Proteins within the plasma membrane perform specific cellular functions of the cell membrane such as cell signaling and cell adhesion [1].
Selection guide for plasma membrane stains
| Readout |
High-performance fluorescent label with stable signal; resistant to photobleaching and pH change
| ||
|---|---|---|---|
| Range |
Binds to N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues on cell membranes
| ||
| Common filter set | DAPI | FITC | TRITC |
| Labels | Invitrogen Alexa Fluor 350 | Invitrogen Alexa Fluor 488 | Invitrogen Alexa Fluor 555 |
| Ex/Em (nm) | 346/442 | 495/519 | 555/565 |
| Signal-to-noise ratio |  | |  |
| Photostability | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
| Fixed cells |
Yes
|
Yes
|
Yes
|
| Fixable |
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
| Format |
5 mg
|
5 mg
|
5 mg
|
| Cat. No. | |||
| Readout |
High-performance fluorescent label with stable signal; resistant to photobleaching and pH change
| |||
|---|---|---|---|---|
| Range |
Binds to N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues on cell membranes
| |||
| Common filter set | Texas Red | Texas Red | Cy5 | Cy5.5 |
| Labels | Invitrogen Alexa Fluor 594 | Invitrogen Alexa Fluor 633 | Invitrogen Alexa Fluor 647 | Invitrogen Alexa Fluor 680 |
| Ex/Em (nm) | 590/617 | 632/647 | 650/668 | 679/702 |
| Signal-to-noise ratio | | | |  |
| Photostability | | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
Yes
|
| Fixed cells |
Yes
|
Yes
|
Yes
|
Yes
|
| Fixable |
Yes
|
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
Imaging
|
| Format |
5 mg
|
5 mg
|
5 mg
|
5 mg
|
| Cat. No. | ||||
| Readout |
High-performance fluorescent label
| ||
|---|---|---|---|
| Target |
Targets plasma membrane
| ||
| Common filter set | FITC | TRITC | Invitrogen Cy5 |
| Labels | Invitrogen CellMask Green | Invitrogen CellMask Orange | Invitrogen CellMask Deep Red |
| Ex/Em (nm) | 522/535 | 554/567 | 649/666 |
| Signal-to-noise ratio | | | |
| Photostability | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
| Fixed cells |
No
|
No
|
No
|
| Fixable |
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
| Format |
100 μL
|
100 μL
|
100 μL
|
| Cat. No. | |||
| Readout |
Expression of fluorescent fusion protein
| ||
|---|---|---|---|
| Target |
Localizes to a plasma membrane-associated kinase
| ||
| Common filter set | DAPI | FITC | TRITC |
| Labels | CFP | GFP | RFP |
| Ex/Em (nm) | 435/485 | 488/520 | 555/584 |
| Signal-to-noise ratio | | | |
| Photostability | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
| Fixed cells |
No
|
No
|
No
|
| Fixable |
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
| Format |
1 mL
|
1 mL
|
1 mL
|
| Cat. No. | |||
| Readout |
High-performance fluorescent label with stable signal; resistant to photobleaching and pH change
| ||
|---|---|---|---|
| Range |
Binds to N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues on cell membranes
| ||
| Common filter set | DAPI | FITC | TRITC |
| Labels | Invitrogen Alexa Fluor 350 | Invitrogen Alexa Fluor 488 | Invitrogen Alexa Fluor 555 |
| Ex/Em (nm) | 346/442 | 495/519 | 555/565 |
| Signal-to-noise ratio | | | |
| Photostability | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
| Fixed cells |
Yes
|
Yes
|
Yes
|
| Fixable |
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
| Format |
5 mg
|
5 mg
|
5 mg
|
| Cat. No. | |||
| Readout |
High-performance fluorescent label with stable signal; resistant to photobleaching and pH change
| |||
|---|---|---|---|---|
| Range |
Binds to N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues on cell membranes
| |||
| Common filter set | Texas Red | Texas Red | Cy5 | Cy5.5 |
| Labels | Invitrogen Alexa Fluor 594 | Invitrogen Alexa Fluor 633 | Invitrogen Alexa Fluor 647 | Invitrogen Alexa Fluor 680 |
| Ex/Em (nm) | 590/617 | 632/647 | 650/668 | 679/702 |
| Signal-to-noise ratio | | | | |
| Photostability | | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
Yes
|
| Fixed cells |
Yes
|
Yes
|
Yes
|
Yes
|
| Fixable |
Yes
|
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
Imaging
|
| Format |
5 mg
|
5 mg
|
5 mg
|
5 mg
|
| Cat. No. | ||||
| Readout |
High-performance fluorescent label
| ||
|---|---|---|---|
| Target |
Targets plasma membrane
| ||
| Common filter set | FITC | TRITC | Invitrogen Cy5 |
| Labels | Invitrogen CellMask Green | Invitrogen CellMask Orange | Invitrogen CellMask Deep Red |
| Ex/Em (nm) | 522/535 | 554/567 | 649/666 |
| Signal-to-noise ratio | | | |
| Photostability | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
| Fixed cells |
No
|
No
|
No
|
| Fixable |
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
| Format |
100 μL
|
100 μL
|
100 μL
|
| Cat. No. | |||
| Readout |
Expression of fluorescent fusion protein
| ||
|---|---|---|---|
| Target |
Localizes to a plasma membrane-associated kinase
| ||
| Common filter set | DAPI | FITC | TRITC |
| Labels | CFP | GFP | RFP |
| Ex/Em (nm) | 435/485 | 488/520 | 555/584 |
| Signal-to-noise ratio | | | |
| Photostability | | | |
| Multiplexing |
Yes
|
Yes
|
Yes
|
| Live cells |
Yes
|
Yes
|
Yes
|
| Fixed cells |
No
|
No
|
No
|
| Fixable |
Yes
|
Yes
|
Yes
|
| Platform |
Imaging
|
Imaging
|
Imaging
|
| Format |
1 mL
|
1 mL
|
1 mL
|
| Cat. No. | |||
Wheat germ agglutinin conjugates for plasma membrane
Wheat germ agglutinin (WGA) is one of the most widely used lectins in cell biology. When conjugated to Alexa Fluor dyes, WGAs are versatile cationic probes for detecting glycoconjugates (i.e., N-acetylglucosamine and N-acetylneuraminic acid (sialic acid) residues) on cell membranes. WGA also serves as a retrograde tracer for neuronal mapping where it will also cross synapses. These fluorescent lectins are applicable in microbiology studies which have been shown to stain gram-positive but not gram-negative bacteria, and stain chitin in fungal cell walls.
WGA conjugates can be used in live and fixed cells. The range of colors provides experimental flexibility for multiplexing and colocalization studies (Figures 1 and 2).
Figure 1. Multicolor fluorescence image of mouse intestine cross section. Filamentous actin prevalent in the brush border was visualized with Alexa Fluor 568 phalloidin. Goblet cell mucus was detected with a blue-fluorescent Alexa Fluor 350 conjugate of wheat germ agglutinin. Nuclei were stained with SYTOX Green nucleic acid stain.
Figure 2. Multicolor fluorescence image of osteosarcoma cells. Fixed and permeabilized osteosarcoma cells simultaneously stained with the fluorescent lectin Alexa Fluor 488 concanavalin A (Con A) and Alexa Fluor 594 wheat germ agglutinin (WGA). Con A selectively binds a-glucopyranosyl residues, whereas WGA selectively binds sialic acid and N-acetylglucosaminyl residues. The nuclei were counterstained with blue-fluorescent Hoechst 33342 nucleic acid stain. The image was acquired using bandpass filter sets appropriate for the Texas Red dye, fluorescein, and DAPI.
CellMask stains for plasma membrane
CellMask Plasma Membrane Stains allow fast and uniform labeling of the cell membrane without the cell-type differences exhibited by lectins. CellMask plasma membrane stains may be used for translocation assays, plasma membrane dynamics, and cell segmentation tool for high-content screening, as well as to stain cellular plasma membranes for standard fluorescence microscopy (Figures 3 and 4).
CellMask plasma membrane stains rapidly stain live cells for 30–90 minutes depending on cell type and experimental conditions. The stains can survive fixation allowing for multiplexing but cannot be permeabilized, so they are not suitable for experiments that also involve probing internal targets.
Workflows are as simple as adding 1X staining solution to cells, incubating for 15–60 minutes, and finished by washing and imaging.
Figure 3. HeLa cell plasma membrane staining using CellMask Green plasma membrane stain. HeLa cells were loaded with 200 nM TMRM in Live Cell Imaging Solution for 30 minutes at 37°C. Cells were washed and then loaded with CellMask Green Plasma Membrane Stain for 15 minutes at room temperature. Images were acquired on a confocal microscope.
Figure 4. U-2OS cell labeling using CellMask Deep Red plasma membrane stain. Human osteosarcoma (U-2OS) cells were labeled with CellMask Deep Red plasma membrane stain, the endoplasmic reticulum-selective ER-Tracker Green dye, mitochondrion-selective MitoTracker Red CMXRos dye, and blue fluorescent NucBlue Live Cell Stain. Labeled cells were imaged in the Live Cell Imaging Solution on the Zeiss 710 point-scanning spectral detection confocal microscope.
Plasma membrane fluorescent fusion proteins
CellLight fluorescent fusion proteins are available as cell membrane labels in live cells to follow the dynamics of cellular behavior. CellLight Plasma Membrane proteins are ready-to-use constructs that express fluorescent fusion proteins bound to the myristoylation/palmitoylation sequence from Lck tyrosine kinase (0.9 kDa) and localize to lipid rafts.
Introducing CellLight fluorescent proteins involves a simple transfection step using the BacMam technology, and they work like cells stains with minimal toxicity or chemical disruption. These plasma membrane proteins are compatible with other fluorescent probes for multiplex analysis in live cells (Figures 5 and 6), or after formaldehyde fixation for colocalization studies.
Learn more about these and other CellLight fluorescent reagents
Figure 5. Live cell imaging with CellLight reagents. HEK-293 cells transduced with CellLight Plasma Membrane-CFP and CellLight Peroxisome-GFP. The following day, live-cell imaging was performed using a 60x objective on a DeltaVision Core microscope and standard CFP/FITC/TRITC filter sets.
Figure 6. U-2OS cell staining. U-2OS cells were transduced with CellLight Plasma Membrane-GFP (green) for 16 hours, followed by labeling with Qtracker 655 Cell Labeling Kit (magenta) for 60 minutes, and HCS NuclearMask Blue (blue). The compiled Z-stack image was acquired on a Zeiss LSM confocal microscope.
Learn more about membrane probes
References
Resources
Molecular Probes Handbook
BioProbes 70 Journal article
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