Toolkit/flow cytometry

flow cytometry

Assay Method·Research·Since 2017

Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.

Summary

Lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min.

Usefulness & Problems

Why this is useful

Flow cytometry was used to study uptake of ceramide analogs by bacteria.; measuring rapid bacterial uptake of ceramide analogs; Flow cytometry is used in the anchor study to immunophenotype peripheral blood lymphocyte populations during autologous stem cell transplantation.; lymphocyte immunophenotyping; cellular composition analysis during ASCT

Source:

Flow cytometry was used to study uptake of ceramide analogs by bacteria.

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measuring rapid bacterial uptake of ceramide analogs

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Flow cytometry is used in the anchor study to immunophenotype peripheral blood lymphocyte populations during autologous stem cell transplantation.

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lymphocyte immunophenotyping

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cellular composition analysis during ASCT

Problem solved

It provides a rapid uptake measurement complementary to microscopy-based localization.; provides uptake readout for ceramide analog interaction with bacteria; It enables measurement of lymphocyte subset dynamics across transplant-associated timepoints.; measuring peripheral blood lymphocyte subsets in multiple myeloma during autologous stem cell transplantation

Source:

It provides a rapid uptake measurement complementary to microscopy-based localization.

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provides uptake readout for ceramide analog interaction with bacteria

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It enables measurement of lymphocyte subset dynamics across transplant-associated timepoints.

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measuring peripheral blood lymphocyte subsets in multiple myeloma during autologous stem cell transplantation

Problem links

measuring peripheral blood lymphocyte subsets in multiple myeloma during autologous stem cell transplantation

Literature

It enables measurement of lymphocyte subset dynamics across transplant-associated timepoints.

Source:

It enables measurement of lymphocyte subset dynamics across transplant-associated timepoints.

provides uptake readout for ceramide analog interaction with bacteria

Literature

It provides a rapid uptake measurement complementary to microscopy-based localization.

Source:

It provides a rapid uptake measurement complementary to microscopy-based localization.

Published Workflows

Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria

2017

Objective: Evaluate antibacterial activity, host-cell compatibility, and bacterial uptake/localization of sphingolipids and ceramide analogs against pathogenic Neisseria.

Why it works: The workflow combines antibacterial potency assays with kinetic, toxicity, and imaging readouts so that active compounds can be linked to rapid bacterial uptake and membrane localization while checking host-cell compatibility.

rapid bacterial uptakebacterial membrane localizationMIC determinationMBC determinationkinetic killing assayflow cytometryconfocal laser scanning microscopydSTORM

Stages

  1. 1.
    Antibacterial activity profiling by MIC and MBC(broad_screen)

    This stage identifies which sphingolipids and ceramide analogs are active against pathogenic Neisseria and distinguishes them from compounds inactive against comparator bacteria.

    Selection: Compounds showing antibacterial activity against Neisseria meningitidis and N. gonorrhoeae in MIC and MBC assays.

  2. 2.
    Kinetic killing characterization(secondary_characterization)

    This stage characterizes the time scale of bactericidal action for an active compound after initial activity has been established.

    Selection: Measure how quickly an active compound kills N. meningitidis.

  3. 3.
    Host-cell toxicity check(confirmatory_validation)

    This stage checks whether antibacterial activity is accompanied by significant toxicity to host cells.

    Selection: Assess whether bactericidal concentrations cause significant host-cell toxicity.

  4. 4.
    Bacterial uptake and membrane localization imaging(functional_characterization)

    This stage provides spatial and temporal evidence that ceramide analogs enter bacteria rapidly and distribute in the bacterial membrane.

    Selection: Visualize uptake timing and membrane distribution of ceramide analogs in bacteria.

Steps

  1. 1.
    Measure MIC and MBC of sphingolipids and ceramide analogs against pathogenic Neisseria and comparator bacteriatested antibacterial ceramide analog

    Identify active compounds and assess organism selectivity.

    Initial potency testing is needed before kinetic, toxicity, and localization follow-up can focus on active compounds.

  2. 2.
    Test killing kinetics of ω-azido-C6-ceramide against Neisseria meningitidisactive hit selected for kinetic follow-up

    Determine how rapidly the active compound kills bacteria.

    Kinetic characterization follows initial activity detection to refine understanding of bactericidal performance.

  3. 3.
    Assess host-cell toxicity of ω-azido-C6-ceramide at a bactericidal concentrationactive antibacterial candidate under safety check

    Check whether bactericidal dosing causes significant host-cell toxicity.

    A host-compatibility check is performed after antibacterial activity is established to evaluate whether the active compound remains usable in a host-relevant context.

  4. 4.
    Measure bacterial uptake of ceramide analogs by flow cytometry and CLSMassays used to quantify and visualize uptake

    Determine whether ceramide analogs are rapidly taken up by bacteria.

    After activity and host-compatibility are established, uptake measurements help connect antibacterial behavior to bacterial association.

  5. 5.
    Visualize membrane distribution of ceramide analogs by CLSM and dSTORMimaging assays used for localization

    Determine spatial distribution of ceramide analogs in the bacterial membrane.

    Localization imaging follows uptake detection to provide higher-resolution spatial evidence about where the compounds accumulate.

Peripheral Lymphocyte Dynamics in the Immune Microenvironment of Multiple Myeloma During Autologous Stem Cell Transplantation

Objective: To prospectively profile peripheral blood lymphocyte dynamics in multiple myeloma during autologous stem cell transplantation.

Why it works: The study follows matched transplant-associated timepoints to compare peripheral lymphocyte states over the course of ASCT.

immune reconstitution during ASCTperipheral blood immunophenotypingflow cytometry

Taxonomy & Function

Primary hierarchy

Technique Branch

Method: A concrete measurement method used to characterize an engineered system.

Target processes

localization

Implementation Constraints

cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationoperating role: sensor

The assay requires detectable lipid labeling and access to flow cytometry instrumentation.; requires flow cytometry instrumentation; It requires peripheral blood samples and a flow-cytometry-based immunophenotyping workflow.; requires peripheral blood immunophenotyping workflow

the abstract does not describe subcellular spatial resolution from flow cytometry alone

Validation

Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication

Supporting Sources

Ranked Claims

Claim 1activitysupports2017Source 1needs review

Short-chain ceramides and ω-azido-C6-ceramide are active against Neisseria meningitidis and Neisseria gonorrhoeae.

Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C6-ceramide were active against Neisseria meningitidis and N. gonorrhoeae
Claim 2host compatibilitysupports2017Source 1needs review

At a bactericidal concentration, ω-azido-C6-ceramide had no significant toxic effect on host cells.

Of note, at a bactericidal concentration, ω-azido-C6-ceramide had no significant toxic effect on host cells.
Claim 3kineticssupports2017Source 1needs review

ω-azido-C6-ceramide killed Neisseria meningitidis within 2 hours at 1× MIC.

Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω-azido-C6-ceramide at 1 X the MIC.
killing time 2 h
Claim 4localizationsupports2017Source 1needs review

CLSM and dSTORM showed homogeneous distribution of ceramide analogs in the bacterial membrane.

CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane.
Claim 5specificitysupports2017Source 1needs review

Short-chain ceramides and ω-azido-C6-ceramide were inactive against Escherichia coli and Staphylococcus aureus.

whereas they were inactive against Escherichia coli and Staphylococcus aureus
Claim 6uptake localizationsupports2017Source 1needs review

Ceramide analogs were rapidly taken up by bacteria within 5 minutes.

Lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min.
uptake time 5 min

Approval Evidence

2 sources1 linked approval claimfirst-pass slug flow-cytometry
Lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min.

Source:

The web research summary states that flow cytometry is used in the anchor study for lymphocyte immunophenotyping and cellular composition analysis.

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uptake localizationsupports

Ceramide analogs were rapidly taken up by bacteria within 5 minutes.

Lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min.

Source:

Comparisons

Source-stated alternatives

The abstract also uses CLSM and dSTORM for localization-focused imaging.; The provided evidence does not name a direct alternative assay in the anchor paper context.

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The abstract also uses CLSM and dSTORM for localization-focused imaging.

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The provided evidence does not name a direct alternative assay in the anchor paper context.

Source-backed strengths

supported detection of rapid uptake within 5 min; supports lymphocyte immunophenotyping in the ASCT setting

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supported detection of rapid uptake within 5 min

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supports lymphocyte immunophenotyping in the ASCT setting

Compared with 3D-dSTORM

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Shared frame: source-stated alternative in extracted literature

Strengths here: supported detection of rapid uptake within 5 min; supports lymphocyte immunophenotyping in the ASCT setting.

Relative tradeoffs: the abstract does not describe subcellular spatial resolution from flow cytometry alone.

Source:

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Compared with confocal laser scanning microscopy

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Shared frame: source-stated alternative in extracted literature

Strengths here: supported detection of rapid uptake within 5 min; supports lymphocyte immunophenotyping in the ASCT setting.

Relative tradeoffs: the abstract does not describe subcellular spatial resolution from flow cytometry alone.

Source:

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Compared with direct stochastic optical reconstruction microscopy

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Shared frame: source-stated alternative in extracted literature

Strengths here: supported detection of rapid uptake within 5 min; supports lymphocyte immunophenotyping in the ASCT setting.

Relative tradeoffs: the abstract does not describe subcellular spatial resolution from flow cytometry alone.

Source:

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Compared with dSTORM

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Shared frame: source-stated alternative in extracted literature

Strengths here: supported detection of rapid uptake within 5 min; supports lymphocyte immunophenotyping in the ASCT setting.

Relative tradeoffs: the abstract does not describe subcellular spatial resolution from flow cytometry alone.

Source:

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Compared with imaging

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Shared frame: source-stated alternative in extracted literature

Strengths here: supported detection of rapid uptake within 5 min; supports lymphocyte immunophenotyping in the ASCT setting.

Relative tradeoffs: the abstract does not describe subcellular spatial resolution from flow cytometry alone.

Source:

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Compared with imaging surveillance

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Shared frame: source-stated alternative in extracted literature

Strengths here: supported detection of rapid uptake within 5 min; supports lymphocyte immunophenotyping in the ASCT setting.

Relative tradeoffs: the abstract does not describe subcellular spatial resolution from flow cytometry alone.

Source:

The abstract also uses CLSM and dSTORM for localization-focused imaging.

Ranked Citations

  1. 1.
    StructuralSource 1Scientific Reports2017Claim 1Claim 2Claim 3

    Extracted from this source document.