HCS/HTS microscopy

[[“”,”Monoculture“,”Co-culture“],[“Working principle“,”Microscopy counting of live or fixed cells involves visualizing, segmentation and automated counting of cells in a prepared sample. This method provides an estimate of the total cell count based on a representative area of the sample. “,”Microscopy cell counting of live or fixed cells involves visualizing, segmenting, and automating the counting of cells in a prepared sample. This method incorporates differential staining techniques to distinguish various cell types or subpopulations within co-cultures. It provides estimates of the total cell count and the counts of specific subpopulations, based on a representative area of the sample.”],[“What is detected“,”Images of cells or nuclei”,”Images of cells or nuclei”],[“Signal type“,”Fluorescence/Brightfield”,”Fluorescence”],[“Platform“,”Automated fluorescence microscope”,”Automated fluorescence microscope”],[“Sensitivity“,”4/4″,”4/4”],[“Throughput“,”High”,”High”],[“End-point/real time“,”End-point / Real-time”,”End-point / Real-time”],[“Multiplexing“,”Yes, for example with Live/Dead or Apoptosis staining kits”,”Yes, for example with Live/Dead or Apoptosis staining kits”],[“Model system“,”Adherent 2D cell cultures or cytospun suspension cells”,”Adherent 2D cell cultures or cytospun suspension cells”]]

The Cell Cycle Analysis Assay is a vital tool for assessing the effects of various compounds and treatments on the cell cycle. This assay provides valuable insights by measuring changes in DNA content, synthesis, and the number of actively dividing cells. It plays a crucial role in understanding how different agents influence the intricate dynamics of the cell cycle, making it an essential component of our research services.

This comprehensive assay involves the following key steps:

• Incorporation of EdU: To detect newly synthesized DNA, we employ a fluorescently labeled nucleoside analog, 5-ethynyl-2′-deoxyuridine (EdU), providing real-time insights into DNA replication.
• Fluorescent Labeling: Cells are carefully labeled with DNA-binding fluorescent dyes, including widely used options like propidium iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), or Hoechst.
• Identification of Actively Dividing Cells: Immunostaining targeting phosphohistone H3 further enhances our capabilities. It allows us to identify cells that are actively undergoing division.
• Fluorescence Microscopy: The labeled cells are subjected to fluorescence microscopy, enabling us to precisely analyze their DNA content and synthesize invaluable data about their cell cycle stage.

Cells navigate through distinct phases during the cell cycle, encompassing G1, S, G2, and M phases. Our approach involves measuring the relative distribution of cells across these phases, providing valuable insights into how specific treatments impact cell cycle progression.

For instance, when assessing the effects of a treatment, we examine the proportion of cells in each phase. A compound that inhibits cell proliferation may lead to an increased presence of cells in the G1 phase. In contrast, a compound that stimulates cell growth could result in a higher proportion of cells in the S phase. Our methodology empowers us to precisely evaluate and quantify these changes using high-throughput microscopy to screen large libraries of compounds or drugs, facilitating a deeper understanding of treatment outcomes.

Assay summary

	DNA staining with one of the intercalating dyes (DAPI, Hoechst, PI, 7-AAD)	DNA staining (Hoeachst) + nucleotide incorporation (EdU) + mitosis marker (pH3 antibody)
Working principle	The method involves the use of fluorescent dyes that bind to DNA, allowing to determine the DNA content of individual cells. This analysis helps identify the different phases of the cell cycle, such as G1, S, G2/M phases.	The method involves incorporation of EdU to estimate the number of cells in the S-phase, followed by the use of fluorescent DNA-binding dye (Hoechst) to identify cells in the G1 and G2 phases based on their DNA content. Additionally, the technique includes staining with a phospho-Histone 3 (pH3) antibody to differentiate cells in the M phase of the cell cycle
What is detected	fluorescence intensity of nuclei	fluorescence intensity of nuclei in 3 different channels
Signal type	fluorescence	fluorescence
Platform	High throughput fluorescence microscope	High throughput fluorescence microscope
Sensitivity	4/4	4/4
Throughput	High	High
End-point/real time	End-point	End-point
Multiplexing	Yes, for example it can be multiplexed with for example Live/Dead assays	Yes, for example it can be multiplexed with Live/Dead assay
Model system	Adherent 2D cell cultures or cytospun suspension cells	Adherent 2D cell cultures or cytospun suspension cells

Example output

The Multiplexed Protein Levels assay is a type of immunostaining procedure used in high-throughput microscopy screening to analyze multiple protein levels in a single sample using sequential immunostaining protocols. This assay is also known as Iterative Indirect Immunofluorescence Imaging (4i) or Cyclic Immunofluorescence (CycIF).

The assay involves the following steps:

• Immunostaining: Cells are labeled with specific antibodies, each tagged with a different fluorophore, allowing for precise detection of multiple proteins of interest.
• Fluorescence Microscopy: The sample is imaged using fluorescence microscopy, and the fluorescence intensity emitted by each fluorophore is measured.
• Iterative Analysis: The strength of this assay lies in its iterative nature. After each round of imaging, the previously used antibodies are either removed or antibody-conjugated fluorophores are bleached. New antibodies are then introduced to enable the analysis of additional proteins of interest.

This versatile assay is widely applied in screening large libraries of compounds or drugs to assess their impact on cellular protein levels. It is a valuable tool for identifying potential candidates in cancer therapies and various other applications, offering a comprehensive view of complex cellular signaling pathways and protein interactions.

Assay aummary

	4i	CycIF
Working principle	This is advanced microscopy technique for analysing multiple protein levels in a single sample through sequential immunostaining. After each round, old antibodies are removed (stripped) for the analysis of additional proteins	This is advanced microscopy technique for analysing multiple protein levels in a single sample through sequential immunostaining. After each round, the fluorophores conjugated to the old antibodies are chemically bleached for the analysis of additional proteins
What is detected	up to 3 different protein targets per each cycle	up to 3 different protein targets per each cycle
Signal type	fluorescence	fluorescence
Platform	Automated fluorescence microscope	Automated fluorescence microscope
Sensitivity	4/4	4/4
Throughput	High	High
End-point/real time	End-point	End-point
Multiplexing	Yes, maximal protein number tested in single sample >40	Yes, maximal protein number tested in single sample 15
Model system	Adherent 2D cell cultures	Adherent 2D cell cultures

Example output

[[“”,”Whole cell morphology“,”Subcellular analysis“],[“Working principle“,”Whole cell morphology analysis utilizes high-resolution microscopy and specific dyes or antibodies that target the cytoplasm or cell membrane, enabling detailed and rapid visualization and quantification of whole cell morphological features, including cell shape, size, circularity, and others.”,”Subcellular analysis employs high-resolution microscopy and specific dyes or antibodies targeting organelle-specific markers. This enables detailed visualization and quantification of subcellular structures, such as nuclei, mitochondria, endoplasmic reticulum lysosomes, lipid droplets and many others. “],[“What is detected“,”whole cells stained with appropriate dyes/antibodies”,”chosen subcellular compartments stained with appropriate dyes/antibodies”],[“Signal type“,”fluorescence/brightfield”,”fluorescence”],[“Platform“,”high content screening microscopy”,”high content screening microscopy”],[“Sensitivity“,”4/4″,”4/4”],[“Throughput“,”High”,”Medium”],[“End-point/real time“,”End-point/real time”,”End-point/real time”],[“Multiplexing“,”Yes”,”Yes”],[“Model system“,”Adherent 2D cell cultures or cytospun suspension cells”,”Adherent 2D cell cultures or cytospun suspension cells”]]

Our state-of-the-art Single-Molecule RNA Fluorescence In Situ Hybridization (smRNA-FISH) service offers an indispensable approach to studying individual gene transcripts within single cells. This sophisticated technique involves the following key steps:

• Cell Cultivation: Cells of interest are cultured within multiwell plates (96 or 384 well) for defined periods, creating an environment conducive to detailed cellular exploration.
• Biochemical Investigations: We afford the flexibility to introduce a spectrum of biochemical interventions. This may involve the application of compounds of interest, thoughtfully aligned with the specific objectives of your research.
• Incubation and Fixation: Following the prescribed incubation period, the fixation process halts cellular processes, ensuring the molecular integrity of the specimens for subsequent analysis.
• Fluorescent DNA Probes: The core of smRNA FISH methodology involves the utilization of custom-designed, fluorescently labeled DNA oligonucleotide probes. The application of multiple probes per mRNA molecule as well as signal amplification methods significantly heightens the sensitivity of the assay, enabling the precise detection of mRNA expression. Our approach accommodates a dynamic range, from individual transcript molecules to several thousand, delivering a comprehensive view of gene expression at the single-cell level.

smRNA-FISH service represents a breakthrough in single-cell gene transcript analysis, shedding light on the intricacies of gene expression dynamics and contributing to a deeper understanding of cellular function and molecular processes. Notably, smRNA-FISH offers distinct advantages compared to traditional RNA-seq and qPCR methods:

• Spatial Mapping: Beyond mere quantification, our technique offers crucial insights into the spatial distribution of transcripts within the cell. This spatial context enriches our comprehension of gene function within distinct cellular locales.
• Multiplexing Capabilities: By fine-tuning the protocol for immunofluorescence staining, we enable the harmonious integration of smRNA-FISH with the concurrent detection of proteins of interest within the same single cell. This multifaceted approach unlocks the ability to scrutinize mRNA and protein expression simultaneously, uncovering intricate correlations between genetic and protein factors.
• Single-Cell Resolution: Unlike bulk RNA-seq techniques, smRNA-FISH provides single-cell resolution, allowing the examination of gene expression at the individual cell level which s crucial for identifying cell-to-cell variations and heterogeneity within a population.
• High Sensitivity: smRNA-FISH is highly sensitive and can detect low-abundance transcripts, making it a valuable tool for studying genes with low expression levels in single cells.
• Complementary Technique: smRNA-FISH can be used in conjunction with single-cell RNA-seq and qPCR to provide comprehensive insights into gene expression at different levels, offering a well-rounded approach to gene expression analysis.

Assay summary

	Stellaris RNA FISH	ViewRNA™ Cell Plus
Working principle	Method employs a set of 48 singly labelled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris RNA FISH Probes bound to target RNA produce fluorescent signals that permit detection of single RNA molecules as diffraction-limited spots by conventional fluorescence microscopy.	Method employs branched DNA amplification technology to amplify signal detection of an RNA transcript. Specific set of approximately 20 short oligonucleotide pairs bind to the target RNA, and signal amplification occurs through a sequential hybridization process.
What is detected	Individual RNA molecules	Individual RNA molecules – signal amplified by increased number of labels associated with each target RNA molecule
Signal type	fluorescence	fluorescence
Platform	Automated fluorescence microscope	Automated fluorescence microscope
Sensitivity	3/4	4/4
Throughput	Medium	Medium
End-point/real time	End-point	End-point
Multiplexing	Up to three different transcripts	Up to three RNA targets simultaneously; a combination of one to two antibodies and a single RNA; or one antibody and two RNA
Model system	Adherent 2D cell cultures	Adherent 2D cell cultures

Example output

Live microscopy is an advanced research tool that allows scientists to study cellular processes in real-time, providing a deeper understanding of dynamic events that might be missed in traditional end-point assays. With the ability to visualize and quantify transient events, as well as fast-kinetic events such as calcium fluxes, and the ability to run long-term time-lapse assays to monitor processes like apoptosis induction, live microscopy offers a wide range of applications for researchers.

Automated live cell imaging systems enhance this technology by enabling continuous monitoring of cells over extended periods of time. This allows researchers to uncover a wealth of cellular dynamics parameters, making live microscopy particularly useful in early-stage drug development. By gaining a deeper understanding of the dynamic nature of cells in-vivo, researchers can identify potential therapeutic targets and develop more effective treatments.

Some specific services that can be provided through live microscopy include:

• Visualization and quantification of transient events, such as dynamic signaling responses
• Study of fast-kinetic events such as calcium fluxes
• Long-term time-lapse assays to monitor processes such as apoptosis induction
• Continuous monitoring of cells over extended periods of time using automated imaging systems to analyze cellular processes such as adhesion, proliferation, invasion, migration and cell to cell interaction

Assay summary

	Live-cell imaging
Working principle	Living cells, whether unstained, stained with fluorescent dyes, or expressing fluorescent proteins, are observed in a high-content screening microscope equipped with an environmental control chamber. This setup enables precise monitoring of cellular dynamics and responses in a controlled en
What is detected	Time lapse images allowing for monitoring of the behavior of cells
Signal type	fluorescence/brightfield
Platform	High throughput fluorescence microscop
Sensitivity	Depends on the type of fluorescence dyes or probes being used
Throughput	Low to Medium
End-point/real time	Real-time
Multiplexing	Possible depends on the probes being used
Model system	Adherent 2D cell mono- and co-cultures