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Facility News
We are slowly entering the holiday season ☀️ ! In theory, we are already in the meteorological summer but we are trapped in a dramatic weather for a while. I know you are all excited to read new papers and protocols along the pool or at the beach 😉so I do hope it will get sunnier in a couple of weeks. If not, you can always count on us to bring a shiny smile on your face and fix your experiments !
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In this month FACS Tips, we are offering a small interlude to our traditional newsletter, take it as a way to muse over technology and what it can - or can't - do for us. Enjoy the read 😊
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Marine Louvet won the mug this month, Congratulations !
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FACS Tips
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The Breakdown - Flow Edition
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Generative AI is an exciting new tool that has touched many facets of our personal and professional lives. Platforms, such as ChatGPT, have become common resources for generating text or images, allowing us to complete tasks efficiently. However, this doesn’t come without its problems. Limitations in the accuracy of responses, along with numerous ethical dilemmas associated with its use, pose challenges for its integration into daily life. These are all much too big of questions to address here; instead, we’ll do what we do best and stick to flow. Nevertheless, it presents an intriguing challenge.
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In this month’s newsletter, we’re taking a departure from our usual overview of an aspect of flow cytometry and drawing inspiration from the GQ YouTube series "The Breakdown." But instead of Alex Honnold breaking down climbing clips from famous movies, we have our Facility Director Jean-François breaking down ChatGPT's answers to some flow cytometry questions. Let's dive in!
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Question: What are the three rules of compensation single stains for flow cytometry?
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ChatGPT: The three rules of compensation single stains for flow cytometry are:
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Single-Stained Controls: Each fluorochrome used in the experiment is individually stained with a single antibody. This means that separate samples are stained with each fluorochrome-conjugated antibody, allowing for the measurement of the emission spectrum of each fluorochrome without interference from other fluorochromes.
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Unstained Control: An unstained control is included to measure the autofluorescence or background signal of the cells. This control provides a baseline reference for setting compensation values by identifying the natural fluorescence of the cells without any specific staining.
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Isotype Control: Isotype controls are stained with antibodies of the same isotype as the experimental antibodies but are specific to irrelevant antigens. These controls help distinguish specific antibody staining from nonspecific background staining, providing a reference for setting compensation values. Adhering to these rules ensures accurate compensation in flow cytometry by providing reference measurements for each fluorochrome and minimizing spectral overlap between detection channels
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Jean-François: Here ChatGPT doesn’t give some rules about single stains and compensations, but kind of a description of some controls used in flow cytometry. It is funny to read in the first paragraph that ChatGPT propose to stain some fluorochromes with an antibody, … and not cells or beads with a fluorochrome-conjugated antibody. Here are the golden rules for single stains and compensations (More info...).
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- Single stain should be as bright or brighter than any sample that the compensation will be applied to.
- Single stains must be treated in the same way as samples (fixation).
- Single stains must match each fluorochrome used – FITC is not the same as GFP, APC is not the same as Alexa 647; One for each antibody coupled to a tandem dye.
- Background fluorescence (autofluorescence) of both the positive and negative populations should be the same
Question: Explain in brief the difference between compensation and spectral unmixing in flow cytometry.
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ChatGPT: Compensation and spectral unmixing are both methods used in flow cytometry to address spectral overlap between fluorophores, but they differ in their approaches:
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Definition: Compensation is a process used to correct for spectral overlap by subtracting the signal from one fluorophore that bleeds into the detection channel of another fluorophore.
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Method: Compensation is typically performed using single-stained and unstained controls to measure the spectral overlap between fluorophores. These measurements are used to calculate compensation matrices, which are then applied to correct fluorescence intensities in multicolor experiments.
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Application: Compensation is commonly used in conventional flow cytometry setups where discrete filters and detectors are used to capture fluorescence signals from different fluorophores.
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Definition: Spectral unmixing is a method used to deconvolute overlapping fluorescence signals by mathematically separating the contributions of each fluorophore based on their unique spectral signatures.
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Method: Spectral unmixing relies on acquiring spectral data for each fluorophore, typically using instruments such as spectral flow cytometers or imaging flow cytometers. Algorithms are then used to computationally separate the individual contributions of each fluorophore from the complex spectral data obtained.
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Application: Spectral unmixing is particularly useful in spectral flow cytometry setups, where the entire emission spectrum of each fluorophore is captured for every particle. It enables more accurate and precise separation of signals compared to compensation, especially in experiments with a high degree of spectral overlap or when using a large number of fluorophores. In summary, compensation corrects for spectral overlap by subtracting fluorescence signals, while spectral unmixing mathematically separates overlapping signals based on their spectral characteristics. Compensation is commonly used in conventional flow cytometry, whereas spectral unmixing is more prevalent in spectral flow cytometry setups.
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Jean-François: The information given by ChatGPT is quite correct, the only non-correct info is the use of unmixing in imaging flow cytometry where compensations are used for managing the spectral overlap in this kind of application.
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Question: Design a 15 color T cell phenotyping panel for the 5 laser BD Fortessa flow cytometer.
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ChatGPT: Designing a 15-color T-cell phenotyping panel for the 5-laser BD Fortessa flow cytometer requires thoughtful selection of fluorophores to maximize signal resolution and minimize spectral overlap. Here's a suggested panel design:
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This panel leverages the available lasers on the BD Fortessa flow cytometer and selects fluorophores with minimal spectral overlap. The chosen markers cover various T-cell subsets, activation markers, and differentiation markers, providing comprehensive phenotyping of T-cells. Additionally, careful consideration has been given to the emission spectra of the fluorophores to ensure optimal detection and resolution of signals on the instrument.
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Jean-François: Definitely, if you need some AI for your panel design, prefer EasyPanel rather than ChatGPT. The suggested panel has several issues: 3 of the antigens are not expressed by T cells, 4 lasers are used for the fluorochrome excitation and not 5, some of the BV dyes are described to be excited by some other lasers than the 405nm, some overlapping fluorochromes combination are proposed like BV421 and Pacific Blue, or BV510 and Pacific Orange… nothing to keep.
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Question: Why is it important to include an Fc blocking step in flow cytometry staining of PBMCs? Answer in brief.
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ChatGPT: Including an Fc blocking step in flow cytometry staining of PBMCs is important to minimize non-specific binding of antibodies to Fc receptors on immune cells, which improves specificity, signal-to-noise ratio, staining efficiency, and consistency of flow cytometry data.
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Jean-François: Overall this is a good answer. Although not all the immune cells express Fc receptors, but B cells, monocytes, macrophages dendritic cells, NK cells and polynuclear cells. It is important to perform the Fc-blocking step prior to staining with antibodies.
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Question: Can I use beads for preparing my fluorescence minus one controls (FMO) in flow cytometry?
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ChatGPT: Yes, you can use beads for preparing your fluorescence minus one (FMO) controls in flow cytometry. FMO controls are essential for properly gating and analyzing your flow cytometry data by providing a reference for the fluorescence signal of each fluorochrome in the absence of one specific antibody.
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Using beads as a substitute for cells in FMO controls can be advantageous in several ways:
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Consistency: Beads provide a consistent and reproducible fluorescence signal, ensuring that the control is reliable and consistent between experiments.
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Uniformity: Beads have a uniform size and shape, which can help ensure consistent staining and fluorescence intensity across different samples.
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Resource Efficiency: Using beads can be more cost-effective and efficient than preparing additional cell samples for each FMO control.
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Flexibility: Beads can be easily multiplexed with different fluorochromes, allowing you to create FMO controls for multiple markers simultaneously.
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When using beads for FMO controls, it's essential to choose beads that are compatible with your flow cytometer and fluorochromes of interest. Additionally, make sure to include appropriate compensation controls to account for any spectral overlap between fluorochromes. Overall, using beads for FMO controls can be a convenient and reliable approach in flow cytometry experiments.
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Jean-François: This one was about to kill me …. NO !!! I cannot use beads for preparing my FMO controls, I must use the same cells as in my final experiment. Fluorescence minus one (FMO) controls are samples stained with all fluorochrome-conjugated antibodies or probes but one of them. They are used to set the gate for the positive population above the background signal observed for the omitted label. FMO controls are crucial when the positive is not clearly separated from the negative. Importantly, FMO controls are affected by both the autofluorescence and the secondary marker expression levels in the cells and therefore need to be same type of cells than the experimental samples and cannot be substituted by e.g. beads or an irrelevant cell line. (More Info...)
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Question: Can flow cytometry be used to count cells?
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ChatGPT: Yes, flow cytometry can be used to count cells. Flow cytometers can measure the number of cells passing through the instrument by detecting individual events (or "particles") as they pass through the laser beam and associated detectors. By analyzing the characteristics of these events, such as their size, granularity, and fluorescence properties, flow cytometry can provide quantitative information about the number of cells present in a sample. This capability makes flow cytometry a valuable tool for cell counting and enumeration in various biological and clinical applications.
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Jean-François: Only the very first sentence of the answer given by ChatGPT is of interest, rest is just some bla-bla … An absolute cell count can be done with some flow cytometers and there are two possibilities: - With some volumetric flow cytometers (like the Beckman Coulter Cytoflex’s or the Cytek Aurora’s instruments in our facility), the volume of cell suspension analysed is measured by the instrument and the cell counting can be automatically calculated. Some of these instruments require a prior calibration for an accurate cell counting. - For all the instruments, counting beads (beads with a pre-determined concentration) can be mixed v/v with cell suspension. By evaluating the ratio of counting beads to cells, the volumetric number of cells may be determined. This approach can be used with any kind of flow cytometer but it is highly sensitive to pipetting errors.
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Question: How does an amine reactive viability dye differ from DAPI for viability in flow cytometry staining?
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ChatGPT: A primary difference between an amine-reactive viability dye and DAPI for viability assessment in flow cytometry staining lies in their mechanisms of action and staining properties:
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Amine-Reactive Viability Dye: These dyes, like propidium iodide (PI) or 7-aminoactinomycin D (7-AAD), work by penetrating compromised cell membranes and binding to nucleic acids, typically DNA, inside non-viable cells. They are impermeable to intact cell membranes and thus selectively stain non-viable cells with compromised membrane integrity.
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DAPI: DAPI is a fluorescent dye that binds specifically to DNA by intercalating between base pairs. It stains all cells, regardless of viability status, as it does not rely on membrane integrity for cell penetration.
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Amine-Reactive Viability Dye: Amine-reactive dyes are commonly used to distinguish between viable and non-viable cells in flow cytometry. They are effective at identifying cells with compromised membrane integrity, which are typically considered non-viable.
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DAPI: DAPI stains all cells, including both viable and non-viable cells, making it unsuitable for specifically assessing cell viability. Instead, it is often used as a nuclear counterstain to identify total cell populations and provide information about cell cycle distribution.
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In summary, amine-reactive viability dyes selectively stain non-viable cells with compromised membrane integrity, while DAPI stains all cells regardless of viability. The choice between the two depends on the specific experimental goals, with amine-reactive dyes being preferred for viability assessment and DAPI being useful for general cell identification and analysis.
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Jean-François: ChatGPT is completely wrong regarding the mechanism of action of both amine-reactive dyes and DAPI.
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Amine-reactive viability dyes are indeed penetrating cells with a compromised membrane, but they do not bind to nucleic acids, they bind to free amine residues on proteins. This is what make them compatible with fixation. Viable cells are also stained with amine-reactive viability dyes as proteins are expressed on the cell membrane, but in a lower extent compared to dead cells having more accessible proteins in their cytoplasm. This is also why viability staining using amine-reactive dyes should be done in a staining buffer without proteins.
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DAPI is not an intercalator even if it stains the DNA by attaching the minor grove of A-T rich sequences. DAPI exhibits around 20-fold enhancement of fluorescence when bind to DNA, this is why it do not require a washing step. DAPI is an impermeant DNA dye and is largely excluded from live cells by competent plasma membranes but can enter a compromised membrane like the ones of dead or fixed cells. (More info...)
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Conclusions from Jean-François:
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From a global point of view, ChatGPT can give some correct information about some very general topics, even if it does not offer the possibility to verify the accuracy of its statements. Regarding more complex information, ChatGPT is completely lost and gives some completely wrong answers. If ChatGPT was a user of the FCF, I would not allow him access to our instruments and would advise him to come and attend the theoretical training which is organized every week. If some users wish to refresh their theoretical knowledge about flow cytometry, they can freely come and attend this training after contacting Jean-François Mayol (jean-francois.mayol@unil.ch).
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