Cell Health and Proliferation

BrdU (bromodeoxyuridine) assays are also a method of labeling proliferating cells. In culture or in vivo, BrdU is a nucleotide analog that can be fed to cells in culture media or via direct injection into an animal to be incorporated into the newly replicating DNA of dividing cells. The BrdU pulse time depends on the mitogen or the rate of cell division. This reveals which of the cells achieved division during the time of the BrdU pulse. While it will not give information about how many times a cell divided or the fate of all of the resultant daughter cells, it is a single cell flow cytometry application and thus additional parameters can be assessed on each cell like DNA content, phenotype of each cell or transcriptional factors and regulators that might have differentially responded to the mitogen.  As this application requires an anti-BrdU antibody, samples must be fixed and permeabilized for the antibody to gain access to the DNA for imaging.

BioLegend now offers Phase-Flow™ BrdU Kits designed for flow cytometric analysis and conveniently contain BrdU pulsing solution, an Anti-BrdU antibody, buffers, DNAse, PBS (with Ca²⁺/Mg²⁺), and dyes for total DNA staining (7-AAD and DAPI). 

Phase Flow™ Alexa Fluor® 647 anti-BrdU Antibody

Ramos cells loaded with BrdU for 1.5 hours (left) or with no load controls (right) stained using the Phase-Flow™ FITC BrdU Kit and DAPI.

Con A-stimulated (3 days) BALB/c mouse splenocytes were fixed and permeabilized with 70% ethanol, then stained with Ki-67 (clone 16A8) Alexa Fluor® 647 (filled histogram) or rat IgG2a, κ Alexa Fluor® 647 isotype control (open histogram).

C57BL/6 mouse frozen intestine section was fixed, permeabilized, and blocked. Then the section was stained with anti-Ki-67 (clone 11F6) Alexa Fluor® 647 (red) and anti-E-cadherin (clone DECMA-1) Alexa Fluor® 594 (green) overnight at 4°C. Nuclei were counterstained with DAPI (blue). The image was captured with a 20X objective.

Detection of Baf3/CCR3 cells viability using Resazurin fluorescence measurement. The increasing cell numbers correlate with the increasing fluorescence.

The Deep Blue Cell Viability™ Kit is formulated to study cell proliferation and quantification. The extent of resazurin reduction and resorufin production is proportional to the number of metabolically active cells (live cells) present in the culture. The Deep Blue Cell Viability™ Kit can facilitate cytotoxicity studies in high-throughput screening, as well as the evaluation of new drugs and chemicals.

Apoptosis is a death process defined by the internal degradation of cellular components without the instigation of a systemic inflammation response. Annexin V is one of the most common probes used to examine early-to-mid stages of apoptosis. Annexin V is a protein that recognizes phosphatidylserine (PS), which is commonly found on the cytosolic side of the membrane in healthy cells. However, in dying or apoptotic cells, PS translocates to the extracellular side of the membrane, exposing it and making it available for binding by Annexin V in a calcium-dependent manner. BioLegend provides several fluorophore-conjugated Annexin V options, as well as an array of impermeant nucleic acid stains to be used for distinguishing apoptotic from necrotic cells, and the Annexin V Binding Buffer required for the calcium-dependent binding of the annexin to PS.

Human T-cell leukemia cell line, Jurkat, treated (top) or non-treated (bottom) with BioLegend's LEAF™ purified anti-human CD95 (clone EOS9.1) mAb (Cat. No. 305704) for 4 hours, then stained with FITC Annexin V Apoptosis Detection Kit with 7-AAD (Cat. No. 640922).

Caspases are an endoprotease family that work as an orchestra of signaling molecules involved in inflammation and apoptosis by cleaving and degrading intracellular components to control the disposal of cellular debris. Dysregulation of caspases underlies diseases like cancer, developmental abnormalities, and autoimmune inflammatory disorders. Caspases integral to the “death cascade” are activated in a middle stage of apoptosis when they can form dimers and be cleaved into large and small subunits of differing function. For example, when a cell begins to enter apoptosis, it releases cytochrome c, a protein involved in mitochondrial respiration, from the intermembrane space. Cytochrome c complexes with Apaf-1 to form an apoptosome that recruits and activates procapase-9, leading to the further activation of caspase-3.

Explore the full interactive poster here…

Activated caspase-3 is involved in the signaling cascade responsible for apoptosis execution. Caspase-3 has many downstream targets to cleave including PARP (poly ADP-ribose polymerase). PARP is involved in repairing nicked DNA and keeping cells viable. Cleavage of PARP inactivates this function and facilitates apoptosis and cell death. This cascade of cytochrome c, caspase-9, caspase-3 and PARP are some of the more popular markers to follow when assessing the progression of apoptosis. A great review of the function of individual signaling molecules involved in apoptosis can be found here.

Other functional probes can also assess live/dead status depending on their cellular vitality. Long-term cell tracking probes like CFSE help to indicate a cell’s health as it relies on its esterase activity. The simplest example of cell vitality, however, is Calcein-AM, Calcein Red-AM and Calcein Violet-AM. These are simple fluorogenic esterase probes that are passively cell-permeant and then subsequently retained by the cell when the probe becomes charged upon conversion. The brighter the signal, the more abundant the esterase activity and the healthier the cell. As cells die, whether by necrosis or apoptosis, the esterase activity will become reduced until no activity remains. These probes can be used to track the cells for as long as the signal persists. However, they are considered short-term trackers since they do not bind to internal proteins like CFSE does, and most cells will eventually start to pump out these probes naturally, or actively in the case of cells that are multidrug resistant (MDR).

Fresh (left) or day-old C57BL/6 splenocytes (right) were stained with 0.01 µM Calcein AM and a cell-impermeant nucleic acid dye, SYTOX™ Red (colored). Black figure represents unstained splenocytes.

Human T-cell leukemia cell line, Jurkat, was treated for five hours with LEAF™ purified anti-CD95 (clone EOS9.1), then stained with an impermeant nucleic acid stain, Annexin V APC, and MitoSpy™ Orange CMTMRos. Data shown was gated on live cells.

HeLa cells were stained with 250 nM of MitoSpy™ Orange CMTMRos (yellow) for 20 minutes, fixed with 4% PFA for ten minutes, and permeabilized with 0.1% Triton X-100 for ten minutes. Then the cells were stained with anti-Cytochrome C Alexa Fluor® 647 (red) and counterstained with DAPI (blue). All three images were merged in the bottom right panel. The image was captured with a 60x objective.

Human T-cell leukemia cell line, Jurkat, was treated for five hours with LEAF™ purified anti-CD95 (clone EOS9.1), then stained with an impermeant nucleic acid stain, Annexin V Alexa Fluor® 488, and MitoSpy™ Red CMXRos. Data shown was gated on live cells.

NIH3T3 cells were stained with 100 nM of MitoSpy™ Red CMXRos (red) for 20 minutes at 37°C, fixed with 1% PFA for ten minutes at room temperature, and permeabilized with 1X True Nuclear™ Perm Buffer for ten minutes at room temperature. Then the cells were stained with Flash Phalloidin™ NIR 647 (green) for 20 minutes at room temperature and counterstained with DAPI (blue). The image was captured with a 60x objective.