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Blog - Immunofluorescence vs. Immunocytochemisty vs. Immunohistochemistry…..What IS the difference?

The term immunofluorescence is often confused with immunocytochemistry and immunohistochemistry. All three of these terms are frequently used interchangeably, and this can lead to confusion when looking to purchase an antibody to use in your microscopy experiment.

Buzz Lightyear, The Walt Disney Company.

Immunofluorescence (IF) refers to the detection method being used (i.e., the use of fluorescent dyes to visualize markers of interest), and has nothing to do with the sample type being used. IF can further be broken down into two categories: direct and indirect.

Direct IF uses a single antibody that is directly conjugated to a fluorescent dye in order to detect the target of interest. Indirect IF uses two antibodies to detect the target of interest. The primary antibody that binds the target is unconjugated, and the secondary antibody that reacts with the primary antibody is conjugated to a fluorescent dye.
What are the pros and cons of each detection method?


Immunocytochemistry (ICC)

Immunocytochemistry is most commonly performed on cells that have been grown in a monolayer in a culture dish or transferred from suspension to a slide for viewing on a microscope. When working with adherent cells, the cells are often grown directly on a coverslip. Depending on the cell type, the cells may not adhere to the coverslip on their own and the coverslips will need to be coated with gelatin or either poly-L-lysine or poly-D-lysine in order to aid in this process. Suspension cells are usually fixed by adding the fixative directly to the cell culture media for approximately 15-20 minutes, washed, and resuspended in buffer and directly placed on a gelatin or poly-L/poly-D-lysine coated slide, and smeared using the edge of another slide or the side of a pipet tip. Suspension cells can also be immobilized to slides using a centrifugation method known as Cytospin™.

In immunocytochemistry, there are generally two types of fixatives used: cross-linking fixatives such as formaldehyde and paraformaldeyhde or organic solvents such as methanol or acetone. The cross-linking fixatives are usually preferred because they tend to preserve the morphology of the cell better by crosslinking proteins and forming a cytoskeletal network. However, this cross-linking of proteins can also mask epitopes and decrease the ability of an antibody to bind to its target, thereby requiring a permeabilization step so the antibody can ultimately access that target.

 

The most common permeabilizing reagent used for immunocytochemistry is Triton X-100, ranging from 0.1-0.5% in PBS, but saponin, digitonin, and Tween-20 can also be used. When using organic solvents as the fixative, a permeabilization step is usually not necessary.

HeLa cells were fixed with 1% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes and blocked with 5% FBS for 30 minutes. Then the cells were intracellular stained with 2.5 μg/ml of Ki-67 (clone Ki-67) Alexa Fluor® 594 (red) in blocking buffer overnight at 4°C and followed by Alexa Fluor® 488 Phalloidin (green) staining for 20 minutes. Nuclei were counterstained with DAPI and are shown in blue. The image was captured with 40x objective
Organic solvents allow for the preservation of cellular architecture, but they also dehydrate the samples, precipitate proteins, denature the protein you are hoping to detect, denature GFP and other fluorescent proteins, and remove lipid-linked proteins and other soluble molecules. Organic solvents work well when staining for cytoskeletal markers.


Immunofluorescence of Clone Nestin 20. Nestin is stained in red and Vimentin in green on methanol fixed mouse embryonic cells. Photo courtesty of R. Goldman, PhD, Northwestern University

You can find our general immunocytochemistry protocols using paraformaldehyde (cross-linking) or methanol (organic solvent) on our website in our Technical Protocol section.

Be sure to also check out our brand new Immunocytochemistry Protocol Video.

Immunohistochemistry (IHC)

Immunohistochemistry is most commonly performed on tissue samples that have either been frozen in a cryopreservation media such as optimal cutting temperature (OCT) compound or fixed in neutral buffered formalin and embedded in paraffin. Immunohistochemistry allows you to visualize your target of interest in tissue samples, while maintaining cellular structure.

Immunohistochemistry- Frozen Tissues (IHC-F)

Preparing frozen tissues for microscopy most often requires snap freezing it in either liquid nitrogen or iso-pentane. Before sectioning the tissue, it is commonly placed in a mold and covered in OCT embedding medium. Once the tissue is embedded, you can move it to the cryostat and begin sectioning. Generally, 5-20 μm sections are made and these are mounted to slides that have been coated with gelatin or poly-L/poly-D-lysine, as with immunocytochemistry.

You can also place the freshly harvested tissue directly into a mold and cover with OCT and then place the mold containing the embedded tissue directly into liquid nitrogen. The tissue block can be stored at -80°C until you are ready for sectioning. Once ready for sectioning, the frozen tissue block can be transferred to the cryostat, sections made, and mounted onto coated slides as above.

Slides can be stored at -80°C up to a year until ready for staining. When ready for use, slides are removed from the freezer and usually fixed with methanol or acetone; therefore no antigen retrieval step is necessary as with formalin fixed paraffin embedded (FFPE) tissues.

C57BL/6 mouse spleen tissue stained for CD4, CD8a, B220, CD169, and F4/80.

View additional details and single stain data.


Immunohistochemistry- Paraffin Embedded (IHC-P)

Paraffin embedding of tissues is ideal for preserving tissue morphology. If maintaining the tissue morphology is absolutely critical due to the markers being stained for, it can be a good idea to perform cardiac perfusion on your animals with phosphate buffered saline, immediately followed by 10% formalin or 4% paraformaldehyde as soon as most of the blood has been flushed from the animal. Once perfusion is complete, the desired tissues and organs are harvested and placed in a fixative and stored on ice until ready to embed.

While perfusion of the animal is recommended since blood is highly autofluorescent, for standard stains where morphology is not as critical, harvested tissues can be placed into a 10% formalin or 4% paraformaldehyde solution until ready to embed.

Prior to starting the paraffin embedding process, the tissue is left in fixative anywhere from 4-48 hours. However, be aware of over-fixing the tissue, as this can cause issues with epitope availability (masking) and decrease antibody binding.

The act of embedding the tissue in paraffin is an involved process and requires one to first dehydrate the tissue with a series of alcohol immersions that increase in concentration. Paraffin is not water soluble, and this ensures that the water that is in the tissue is slowly replaced by the alcohol. As paraffin is not soluble in alcohol either, the next step in the process is called clearing, and this refers to the solvents that are used in this process. The alcohol that has replaced the water and dehydrated the tissue is replaced by an organic solvent, most commonly xylene, which makes the tissue receptive to the paraffin. As the name implies, clearing also makes the tissue clear or transparent. The tissue is now ready to be embedded in warm paraffin, which fills the space previously occupied by the water. Once the paraffin cools, the tissue hardens and is ready to be sectioned using a microtome. Sections of approximately 5-8 μm are cut and mounted on glass slides. As most antibody staining solutions are aqueous, you now need to remove the paraffin and replace the water that you just removed! In order to do this, you basically repeat the dehydration and clearing steps in reverse. The slides are deparaffinized by passing through xylene and then decreasing amounts of alcohol.
Antigen Retrieval Methods

Due to the crosslinking fixatives used in the paraffin embedding protocol, antibodies may not be able to bind due to epitope masking. In order to make the binding epitope available, or unmask it, a process called antigen retrieval needs to be performed. Antigen retrieval reverses the crosslinked proteins, thereby allowing the antibody access to the epitope it needs to bind to. There are generally two types of antigen retrieval methods: Heat Induced Epitope Retrieval (HIER) and Proteolytic Induced Epitope Retrieval (PIER).

Heat Induced Epitope Retrieval (HIER)

HIER is the most common type of epitope retrieval that is used as the protocol is more convenient than PIER. There are several important factors that need to be tested and optimized in order to find the appropriate conditions for the antigen you are working with. The incubation time, pH and components of buffer, and temperature are all things to take into account when developing your protocol. The most common buffers used in HIER are either a low pH (~6.0) sodium citrate or a high pH (~9.0) Tris/EDTA used at a temperature of ~95-100°C for 10 min. Buffer conditions, temperatures, and incubation times should be carefully optimized, and may need to be determined for each experiment depending on the antigen.

Human paraffin-embedded colon tissue slices were prepared with a standard protocol of deparaffinization and rehydration. Antigen retrieval was done with Citrate Buffer, pH 6.0 at 95°C for 40 minutes. Then, the tissue was stained with 10 μg/mL of Alexa Fluor® 647 anti-Vimentin (clone O91D3) antibody (green) and 10μg/mL of Alexa Fluor® 594 anti-CD66d/e (clone 308/3-3) antibody (red) over night at 4°C. Nuclei were counterstained with DAPI (blue).

Proteolytic Induced Epitope Retrieval (PIER)

Some antigens may require a little harsher method in order to unmask the epitope and make it available for antibody binding. PIER uses enzymes in order to digest the protein cross-links that were formed during fixation. The most common proteolytic enzymes used are trypsin, pronase, and Proteinase K. As with HIER, conditions need to be tested in order to find the optimal conditions for antigen retrieval. The incubation time, temperature, and concentration of proteolytic enzyme used are all critical.

Hopefully after reading this blog, you have a better understanding of the differences between IF, ICC, and IHC. The take home message is that immunofluorescence is a detection method, and has nothing to do with the type of samples you may be working with.

Star Wars Episode VII: The Last Jedi.
The Walt Disney Company.

BioLegend is aware of the confusion with the terminology, and we are currently working on updating all of our technical data sheets (TDS) to reflect whether a product can be used in ICC, IHC-F, or IHC-P. The term immunofluorescence (IF) will eventually be removed from all TDS’s. We always encourage checking the TDS to see which applications we’ve quality tested or validated and what has been reported in the literature. You can find more reagents, tips, and protocols with our Microscopy Webpage. As always, feel free to contact our tech support group at tech@biolegend.com if you have any questions!!

Contributed by Kellie Johnson.



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Video - Immunocytochemistry Protocol Step-by-Step
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Blog - 5 Reasons to Use Veri-Cells™
Reliable, consistent experimental controls are paramount for validating reagent performance in biological research. Our Veri-Cells™ are lyophilized cells designed to be used for flow cytometry antibody validation. While they're useful as controls in multi-center or longitudinal studies to monitor assay consistency, Veri-Cells™ would be useful for anyone doing flow cytometry research! In this blog, we'll outline 5 reasons why you should use Veri-Cells™, and what makes them the right product for you!

Set proper experimental controls, you must. Yoda knows. The Walt Disney Company.

1) Stability

Our Veri-Cells™ have excellent long-term stability - two years in its lyophilized form, and five days post-reconstitution. Our Veri-Cells™ are available in smaller sizes so you don't need to reconstitute larger batches at once and can keep them lyophilized for long-term storage and use in the future.

2) Validity

Veri-Cells™ PBMC and Veri-Cells™ Leukocytes have been validated for expression of over 150 surface markers using our LEGENDScreen™ Human PE Kit. Additionally, we have validated intracellular expression of commonly examined markers Granzyme B, Perforin, FOXP3, and Helios. You can check out the listing of validated markers, as well as additional stability information and representative data, on the Veri-Cells™ section of our flow cytometry control page.


3) Accessibility, convenience

Veri-Cells™ are perfect for those who may have difficulty accessing human blood, or do not have the time to enrich blood cells and freeze them. We deliver our products overnight within US and Canada (and some European destinations from our UK location). Once you order your Veri-Cells™, they can arrive in your lab as early as the next morning. You can purchase them in bulk, hold onto the lyophilized product, and just reconstitute a vial of cells when necessary.

4) Variety

We offer an ever-expanding variety of Veri-Cells™ products, depending on the target antigens you want to observe. This allows you to forego setting up cumbersome stimulation experiments you may not have performed on your own, and obtain control samples containing markers/cells that are otherwise difficult to detect in normal blood, such as CD34+ hematopoietic stem cells.

Examples of Veri-Cells™ products designed to serve as controls to detect markers that normally require stimulation (Veri-Cells™ Phospho PBMC (MAPK/ERK Pathway), left plots) and are difficult to detect in normal human blood (Veri-Cells™ CD34 PBMC, right plots). Check out their respective webpages to learn more about these products!

Don't see cells and/or desired pre-treatment conditions youre looking for? We're open to taking on custom projects to generate a lyophilized cell control to fit your needs. If you're interested, contact us at tech@biolegend.com for more details.

5) Consistency, reliability

We analyze every lot of Veri-Cells™ produced for a select number of phenotyping markers to ensure that the cell populations/marker staining is within the expected frequency. Observed experimental values from our QC testing are listed on their certificates of analyses, so you have a point of reference for the vial of cells you have on-hand. Of course, this is subject to some degree of variability (e.g. clone of antibody being used to stain, gating for analysis, etc.), but you'll have peace of mind knowing that the vial you received has been tested in comparison to a gold standard to ensure relative consistency. As with all of our reagents, our Veri-Cells™ are also backed by our 100% satisfaction guarantee.

Two Veri-Cells™ PBMC lots reconstituted and stained on different days, and ran on separate flow cytometers. Gating is based on unstained Veri-Cells™ PBMC controls ran simultaneously for each run. The CD4 vs. CD8 plot is on CD3+CD19- gated cells, and the histogram for CD56, CD16 is plotted on CD3-CD19- cells.

Contributed by Kenta Yamamoto, PhD.


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Podcast Episode - Skull Microtunnels, Gut Bacteria, and Fantasy Scientist Drafts
Check out our new podcast to learn about fast-travel for cells in the skull, gut bacteria influence on the body, and whether we're starting a fantasy scientist draft league!

Topics

Microtunnels in the Skull (2:11-10:04)
Russia Is Opening A "Jurassic Park-Style" Research Lab (10:05-19:45)
The Fantasy Scientist Draft (22:05-26:25)
Gut Bacteria, Bullying, Junk Food, and You! (26:26-42:20)
God of War Shout-out (46:52)
Successful Placebo Pills (47:48-52:20)

Keywords: Immunology, podcast, brain, skull, marrow, microtunnels, extinction, Jurassic park, dodo, wooly mammoth, dna, fantasy football, microbiota, gut bacteria, memory, bullying, junk food, diet, psychobiotics, elderberries, Placebo Effect, honest placebo, IBD, inflammatory bowel disease


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Blog - The Gut Microbiota in Control of Autoimmunity
The microbiota is composed of bacteria, fungi, viruses, and other microbial and eukaryotic species which reside in different tissues and biofluids including gut, skin, lung, saliva, oral cavity, kidneys, and reproductive tracts. Of these, the gastrointestinal tract is home to the largest community of bacteria which is estimated to be about 100 trillion cells in humans and outnumbers the host's cells ten-fold.

The host maintains a homeostatic relationship with the microbiota by minimizing the contact between microorganisms and the gut epithelial cell surface. The intestinal epithelium provides a physical barrier between the intestinal lumen and the inside of the body. Goblet cells secrete mucins forming a mucus layer above the epithelial cell layer. In addition, epithelial cells are potent producers of antimicrobial peptides and cytokines.


By Peter Andrey Smith, Boston Globe
Recent studies have shown that microbiota play an important role in controlling different aspects of host physiology including immune responses. Advances in next-generation sequencing led to an understanding of the diversity of microbiota species in health and disease. These studies have shown that each individual harbors a unique microbiome consisting of genes derived from an average of 150 individual bacterial species, each of which includes about 200 strains.

The influence of microbiota on development and maturation of the immune system was demonstrated on germ-free mice which exhibit an underdeveloped immune system associated with reduction in size and number of secondary immune organs including Peyer's patches, altered crypt structure and reduced mucus production by goblet cells. Germ-free mice were further utilized as a model to study the effect of bacteria strains on development of the immune system.
Gut commensals can be subdivided into two major categories, inflammatory and immunoregulatory, depending on their effect on the immune system. Figure 1 describes how the anti-inflammatory or pro-inflammatory responses in the steady state, infection, or inflammation can influence disease formation and progression.

In the steady state, CD103+ mucosal DC cells sample commensal bacteria using pattern-recognition receptors (PRRs), like Toll-Like Receptors. Then CD103+ mucosal cells induce differentiation of Treg cells. In turn, Treg cells block the activation of T helper cells, like Th17 and Tfh cells, and help B cells to produce IgA, restricting the growth or inflammatory effects of commensals. Bacteria strains that have shown immunoregulatory capabilities include Clostridium, Bacteroides fragilis, and Bifidobacterium infantis.

Figure 1. The anti-inflammatory or pro-inflammatory responses
in the steady state or during infection or inflammation.

On the other hand, potentially proinflammatory bacteria strains, like Segmented Filamentous Bacteria (SFB) and Helicobacter hepaticus, can induce differentiation of inflammatory dendritic cells, resulting in expansion of Th1 and Th17 cells. This can lead to the induction of autoimmune diseases within the gut tissue, such as inflammatory bowel disease, Crohn's disease, and colitis, or in distal sites likely due to molecular mimicry between the bacterial and self antigens. Table 1 shows a correlation between some non-gut autoimmune diseases and microbiota.


Table 1. Alterations observed in the microbiota in non-intestinal autoimmune diseases.

Host microbiota are established by many factors including age of the host, genetics, antibiotic usage, sanitary living conditions, and dietary habits. Diet is one of the important factors affecting the composition of microbiota in humans, and therefore, can directly influence the function of the immune system.

Commensals harvest energy from non-digestable dietary components such as starch, cellulose, or xylans providing an additional source of energy for the host. End products of this process include Short Chain Fatty Acids (SCFAs) such as butyrate, propionate, acetate and pentanoate acids. SFCAs provide an energy source for colonic epithelial cells increasing the intestinal epithelial barrier integrity and influence the Treg differentiation as well as T cell cytokine production. Another regulator of lymphocyte response produced by bacteria or triggered upon host cell interaction with bacteria includes Retinoic acid (RA), plant-derived flavonoids and glucosinolates, and gluten.


Comic by Dzu-Doodles
In conclusion, we have discussed an interplay between the gut commensals and the immune system. As you can see, the changes in microbial composition affects host immunity and influences the formation and/or outcome of autoimmunity. A better understanding of human microbiome and its interaction with the host is important for understanding the pathophysiology of human diseases and developing more effective disease treatments.

Contributed by Ekaterina Zvezdova, PhD.

References:
  1. Role of the microbiota in immunity and inflammation
  2. Immune-microbiota interactions in health and disease
  3. The interplay between the gut microbiota and the immune system
  4. The microbiome and innate immunity


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Blog - Advancements in using iNKTs for Cancer Immunotherapy
Immunotherapy harnesses the body's own immune system to provide anti-tumor responses and fight cancer. There are several immunotherapy-based treatments that have been approved for clinical use and many others that are still being developed. Previously, on the blog, we've looked at immune checkpoint inhibitors and CAR-T cells. In this blog, we'll focus on research being done to develop immunotherapies that exploit a specific immune cell - Invariant NKT cells (iNKTs).

What are iNKT cells?

Like their name suggests, NKT cells have characteristics of NK and T cells and express both a T cell receptor complex and many canonical NK cell markers. While there are a few different subsets of NKT cells, for the purpose of this blog, we'll focus on iNKTs. iNKT cells express an invariant TCR with a limited repertoire that responds to the presentation of glycolipids by a class-I related cell surface molecule, CD1d. Upon stimulation, they are able to release a broad range of cytokines to elicit adaptive and innate immune responses, activate other immune cell populations, and induce dendritic cell maturation. Learn more about NKT cell development and function on our NK and NKT cell webpage!

 

iNKT cells can recognize tumor cells both indirectly and directly, either by recognizing CD1d on antigen-presenting cells in the tumor microenvironment or CD1d expressed by the tumor cell. In addition to cytokine release, iNKT cells secrete cytotoxic molecules including perforin, granzymes, and Fas ligand which can cause cytolysis of tumor cells that express CD1d. Because iNKT cells bridge both arms of the immune system, are cytotoxic, and are able to produce inflammatory cytokines, they are very attractive targets for developing immunotherapies. In fact, there have been several studies correlating increased frequency/activity of iNKT cells with overall survival in a number of different tumor types (reviewed in 6, 9). A role for iNKTs in tumor clearance has been well-documented in mouse models, and adoptive transfer of iNKTs using mouse tumor models also suggests that iNKTs can contribute to an anti-tumor response (1).

iNKT cells can recognize CD1d expressed on Antigen Presenting Cells (APC) or Tumor-Associated Macrophages (TAMs). In response, they can release cytokines to activate other immune cells like NK cells, and release cytotoxic molecules for direct killing of the tumor cells.

Stimulation with α-GalCer

Functionally, iNKT cells respond to the α-GalCer glycolipid bound to CD1d. Initial clinical trials concentrated on administration of α-GalCer to stimulate iNKT cells and increase cytokine production (3, 5, 7). Though administration of α-GalCer does not seem to have any major toxicity-related effects, one caveat to this method is that it relies on the quality and the quantity of the existing pool of iNKTs. Upon repeated stimulation with α-GalCer, there are fewer iNKT cells in circulation and cytokine production diminishes due to anergy of iNKTs. A number of strategies may help to circumvent these issues including using analogs of α-GalCer, directing their delivery by incorporating glycolipids into nanovectors, and optimizing the routes and schedule of administration of α-GalCer.

Mouse models have suggested that adoptive transfer of α-GalCer loaded-APCs elicits a stronger immune response (reviewed in 6, 9). As such, in lieu of providing exogenous α-GalCer, some Phase I clinical trials have focused on adoptive transfer of monocyte-derived DCs which have been incubated with α-GalCer in culture. These treatments have resulted in increased numbers of activated iNKTs and downstream activation of B cells, T cells and NK cells. It is worth noting that while these studies have shown expansion of the iNKT cell populations, they typically show robust immunological responses in patients that have "normal" iNKT cell numbers prior to treatment.


α-galactosylceramide is a synthetic glycolipid originally derived from a marine sponge, Agelas mauritianus (pictured above) and can be used as a potent activator of iNKT cells.

Adoptive Transfer of iNKT cells

As the treatments described thus far are dependent on the number of existing iNKT cells, another complementary approach to exploiting iNKTs for immunotherapy involves directly transferring activated iNKT cells into patients. Initial studies found that there were no toxic effects due to the adoptive transfer of stimulated iNKTs but the expansion of the iNKT cell population was transient. For these studies, PBMCs are typically stimulated with α-GalCer in culture; however, newer studies have utilized an antibody directed against the TCR Vα24-Jα18 (clone 6B11) to isolate iNKTs prior to stimulation and transfer, resulting in higher purity and a larger number of iNKTs that can be transferred (2). While these studies are promising, additional studies are necessary to optimize these treatment strategies to demonstrate efficacy.

CAR-iNKT Therapies?

Chimeric-antigen receptor (CAR) T cell based immunotherapies have provided promising therapeutic options and many ongoing studies are working to develop CAR-iNKT-based therapies. CAR-iNKTs may be particularly exciting because iNKT cells have intrinsic anti-tumor responses through recognition of CD1d. Addition of a second recombinant TCR may give the cells multiple mechanisms for tumor recognition. Current studies have demonstrated that CARs can be successfully transduced into iNKTs using either a TCR that recognizes GD2 in neuroblastoma models or a CD19-specific CAR in mouse B cell lymphoma models (4, 8). Two of the major hurdles associated with CAR-T cell studies are tumor homing and graft vs. host disease (GvHD). Interestingly, in mouse models, CAR-iNKTs do not cause GvHD to the same extent as CAR-T cells and these studies have also suggested that tumor homing may be improved.

Comic by: Pedromics

Immunotherapies are constantly evolving and improving, and while many of the current therapies focus on manipulation of T cells, utilizing iNKTs may provide added benefits to anti-tumor responses. The ability of iNKT cells to both directly and indirectly contribute to tumor killing suggests that they may be exploited to treat a wide-range of tumors. While it is important to note the potential caveats associated with these strategies, there is promising research finding new ways to harness the power of iNKTs to fight cancer!

 

Contributed by Kelsey Swartz, PhD.

 

References:
  1. Bellone M, et al. 2010. PLoS One. 5(1):e8646.
  2. Exley MA, et al. 2008. Eur J Immunol. 20:1756.
  3. Fujii S, et al. 2002. Nature Immunol. 3:867-74.
  4. Heczey A, et al. 2014. Blood. 124(18):2824-33.
  5. Kawano T, et al. 1997. Science. 278:1626-9.
  6. King L, et al. 2018. Frontiers in Immunology. 9:1519.
  7. Parekh VV, et al. 2005. J Clin Invest. 115: 2572-83.
  8. Tian G, et al. 2016. J Clin Invest. 126 (6):2341-55.
  9. Wolf B, et al. 2018. Frontiers in Immunology. 9:384.


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Blog - A Guide To Establishing Baseline PMT Voltages
In any lab, reproducibility is crucial for long term success. Since many flow cytometry instruments are highly mutable, reproducibility can become a challenge. Unknowingly having a filter set swapped out or an insufficiently powered laser can be detrimental. Therefore, it is vital to ensure your highly tuned instrument is performing at its best, so you have no doubts about the data after you run your sample.

The Lord of The Rings, New Line Cinema.

To assure that instruments are performing optimally over a long period of time, beads with embedded fluorescence are often used to find voltages that both minimize electronic noise and maintain the fluorescence signal in a linear range for each detector. Typically baseline assessments of the specific channels are performed when any major change occurs to the instrument itself. A common misconception is that the voltages automatically assigned by these beads are suitable for your particular experiment. This is not the case, as these beads are used to follow an instrument's health and should not be utilized to establish experiment-specific PMT voltages. If you were to use the baseline voltages for your multiple color assays, you would have optimized your assay for beads rather than your cells.

Comic by Sidney Harris.

There are multiple ways to assess these baseline voltages. Some instruments use specific beads called Cytometer Setup and Tracking (CS&T) beads to determine baseline voltages known as "application settings". These beads are composed of three different fluorescent intensities ranging from dim to bright. After running CS&T baseline, the bead-specific software calculates the standard deviation of electronic noise. For these beads, the PMT voltage that is selected is 10 times greater than the electronic noise for each channel.
If your current setup does not use CS&T beads or you would like to perform this type of baseline assessment manually, you're in luck. There are several other methods for calculating these values using fluorescent beads. In Stephen Perfetto's 2012 Nature Protocol paper, the authors validated a method where you run both a bead that has multiple fluorescent intensities and an unstained non-fluorescent compensation bead in 50 V increments from 350 to 800 V. The unstained compensation beads are meant to represent the background noise. Once these runs have been completed, you then plot the negative population and the positive populations for each voltage increment. The baseline voltage range for each channel is then selected based off the voltage increment that has the greatest separation between the negative and positive populations (based on MFI or SI), while the linearity (the MFI between the different fluorescent intensities) remains consistent. While this protocol is highly practical, it does require a large amount of time and only delivers a baseline range rather than an absolute baseline that the CS&T beads and software provide.

Alternatively, if you are interested in a method that is a bit less cumbersome, you could instead establish the baseline voltages off the inflection point of Rainbow Calibration Particles, peak 2. Similar to the Perfetto method, this baseline assessment would still require you to acquire the beads at varying voltages. But, instead of determining the maximum separation between negative and positive peaks, you would only need to collect the CV of the dim population. For each voltage increment, both the CV and PMT voltage are recorded. These data points would then be plotted for each specific detector. An inflection point at which the CV remains consistent would serve as the baseline voltage for each specific channel. It's worth noting that this method relies entirely on the dim population of beads.

Dexter's Laboratory, Cartoon Network.

While there are tradeoffs to all three of the methods mentioned above, each provides a unique way to standardize your flow cytometer. These baseline settings will help monitor for any changes or issues that might occur due to problems associated with the laser, electronic noise, filters, and PMTs. Any swings in these voltages should be investigated further. Lastly, it's important to remember that these baseline settings are not meant to be experiment-specific. However, following these protocols will set you on the right track to producing reliable, consistent data with your flow cytometer. Stay tuned for a follow-up blog that delves into voltage and PMT settings for experiments.

 

References:
  1. Quality assurance for polychromatic flow cytometry using a suite of calibration beads 
  2. Flow cytometry controls, instrument setup, and the determination of positivity 
Contributed by Sean Cosgriff.


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Blog - DNA Dyes in Flow Cytometry and Microscopy

"Oh well, Mr. DNA, where did you come from?"
-John Hammond, Jurassic Park. Universal Studios.


As your introductory science classes taught you, DNA is the blueprint for life. And, it tends to be an important focal point in many researchers' work. In this blog, we'll go over how DNA-binding dyes work and how they can be used in flow cytometry and microscopy applications.

Why are we looking for DNA?

DNA can provide lots of insight into the inner workings of a cell. In flow cytometry, we can use DNA-binding dyes to help assess the cell's health. If a dye is not cell-permeant (more on this later), it's excluded by a healthy and intact cell membrane. If that membrane is damaged or the cell is going through apoptosis, the dye can then finds its way to the nucleus and stain DNA. While some people will rely solely on forward and side scatter profiles to assess viability, this is not always reliable and may not include cells in the early stages of apoptosis that have not shrunken down as they die and become debris.

Versatile Helix NP™ Dyes

C57BL/6 mouse thymus cells were fixed using 70% chilled ethanol. The cells were incubated for one hour at -20°C, washed, then stained with Helix NP™ NIR at 5 μM. HeLa cells were fixed, permeabilized, and blocked. Then the cells were intracellularly stained with Alexa Fluor® 488 anti-Cytokeratin (pan reactive)(green) antibody followed by Helix NP™ NIR (red)
DNA dyes can even show you what stage of the cell cycle the cell is currently in. Due to the stoichiometric binding of nucleic acid stains, cells in the G2 or M phase have double the DNA content of a normal cell before dividing and thus, stain with much higher levels of Helix NP™ NIR and have a higher mean fluorescence intensity. DRAQ5™ and CytoPhase™ Violet can be used in a similar manner. In microscopy, the applications for DNA dyes are a little different. DNA dyes are typically used to counterstain cells or tissues. This gives better contextual and localization clues to proteins you've stained with antibodies.

Traits of DNA Dyes

Some of the important traits to understand about your DNA dye include permeability and their mechanism of action. Refer to our handy chart to keep track of these characteristics.

Permeability

Permeability is a simple enough concept: can the dye get into the cell to bind DNA if the membrane is intact? Permeability will define the type of assay your dye is useful for. If it's cell-permeant, it's less suitable for a viability assay since it will get into every cell regardless of its health status. Instead, it will work better to give you an idea of a total cell count or cell cycle status. If a dye is not cell-permeant, then it can be helpful in assessing viability. For our Helix NP™ dyes, NP indicates they're "Not cell-Permeant" so they're helpful assessors of cell health. Similarly, DRAQ7™ and Propidium Iodide are also excluded by healthy cells with intact membranes. Other dyes like DAPI and 7-AAD are classically considered not to be cell-permeant. However, they are actually semi-permeant, meaning at high concentrations, they'll get through a live cell's membrane and stain, but to a lesser degree than a dead cell would.

Mechanisms of Action

It can also be helpful to understand how these dyes are binding to DNA. Different interactions with DNA have different affinities. The higher the affinity, the tighter the binding and the more easily you can resolve DNA and see more than just a haze around the nucleus. We'll be focusing on dyes that bind the minor groove and intercalators. The minor groove is the narrower of the two grooves formed by the double helix structure of DNA. Minor groove binders generally have a lower affinity than intercalating dyes. Minor groove binders may be a little less flexible as they must follow the groove as it twists around the axis, coming into contact with the edges of base pairs. Binding typically occurs through non-covalent means (i.e., hydrogen bonding of the probe to base pairs).

 

DNA intercalators actually insert or "intercalate" non-covalently between two sets of adjacent base pairs, causing them to separate and create a pocket for the dye. A part of dye's structure, such as a hydrophobic aromatic ring that bears resemblance to a ring of bases in DNA, can insert itself between sets of base pairs. This can actually cause the DNA to become distorted. Bis-intercalators contain two intercalating moieties connected by a linker that interacts with a groove. As such, they have an even higher affinity than single intercalators and have even been used to image single strands of DNA. Minor groove binders and intercalators can increase in fluorescence due to conformation changes upon binding DNA. It is important to notice that most DNA-binding dyes can be dislodged if the samples are fixed or permeabilized. This is particularly important in scenarios where organic solvents with DNA-denaturing properties are used. For example, methanol is commonly used to help solubilize paraformaldehyde in certain fixation solutions. This can lead to false positives and negatives, making it harder to interpret your data. Alternatively, you can analyze viability with Zombie Dyes, which bind amines on proteins and are more well-suited for fixation and permeabilization conditions.
Hopefully, now you have a better understanding of how DNA-binding dyes work and which one you should choose for your applications. If you want to learn more about DNA dyes, other chemical probes, and their applications, check out our Cell Health and Proliferation Webpage. And if you still have questions, contact our tech support group.

Contributed by Ken Lau, PhD.

So that’s where Spider-Man got that catchphrase.
Comic by the Amoeba Sisters.




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