4
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`0 g
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`BOS
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`32ND
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`CNAT CIET
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`AN E 10
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`14, 2017
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`NES CONVENTION CEgy
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`TFS1067
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`1
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`several drugs showing promise after
`testing with standard
`Despite
`including extensive
`m vitro assays,
`cell
`line panels, many tumors
`the clinical
`respond
`these drugs at
`trial stage. Thus, despite
`fail
`to
`to
`the inability of pre-clinical
`huge monetary
`investment
`trials,
`in
`to predict success is hampering efforts to progress cancer
`models
`and to personalize cancer chemotherapy. Cell
`treatment
`lines have
`been the workhorse of cancer
`research for decades;
`however,
`they
`tumor heterogeneity or patient
`do not
`represent
`variability.
`There is
`to carry out pre-clinical
`a need for
`testing in order to
`a better model
`of success in clinical
`trials, which would in
`give greater chance
`turn
`reduction of wasted
`mean benefit
`for more patients and an overall
`funds.
`
`To address this need for more clinically relevant models,
`the use
`of
`primary
`is becoming more
`cell cultures
`derived
`from patient
`tumors
`desirablea2ßlintegrinhi/CD133)
`and committed basal cells (CB)
`(a2(31integrinl°). Alongside this novel
`imaging technique we used
`labelling of surface markers
`fluorescent
`to confirm cell
`identity.
`
`use ptychography to carry out
`Our aim is
`real-time analysis of
`to
`cell
`response to drug treatments,
`using docetaxel
`the standard of
`and comparator. A full characterization af
`care treatment
`types of
`all
`patient-derived tumor
`cells, alongside analysis
`their
`response to
`of
`current and novel drugs will allow assessment
`of detailed biological
`the drugs tested as well
`identification of resistant
`effects of
`cells. This could lead to patient
`cells becoming part of
`the drug
`development pipeline, which will ultimately result
`targeted
`and
`in
`take into account
`patient
`stratified
`therapies
`that
`intra- and inter-
`tumor heterogeneity.
`
`as
`
`as
`
`n8alys2i
`
`of Variability and Programmed Cell Death in
`Cyanobacteria Using Imaging Flow Cytometry
`Veronika Dashkoval,
`Ivan Vorobjev2, Natasha S
`Bartenevaa
`
`I
`
`W
`
`that
`
`for Life Sciences, National Laboratory Astana,
`'Center
`Nazarbayev University, Astana, Kazakhstan, 2Schoolof
`Science and Technology, Nazarbayev University, Astana,
`3Program in Molecular and Cellular Medicine,
`Kazakhstan,
`Boston Children's Hospital, Harvard Medical School, Boston,
`MA, United States
`Prokaryotic cyanobacteria are photosynthetic prokaryotes
`are responsible for production of approximately 25% of all
`carbohydrates on Earth (Scanlan, West, 2012) and are also believed
`the chloroplasts in eukaryotic phytoplankton
`an ancestor
`of
`to
`be
`and plants. The evolution of nitrogen fixation along with oxygenic
`has promoted development
`photosynthesis
`of cell specialization,
`in morphologically
`and functionally diverse cells within
`resulting
`populations of nitrogen-fixing filamentous cyanobacteria. Moreover,
`programmed cell death (PCD),
`responsible for
`a programmed
`of morphological
`in multicellular
`sequence
`changes
`organisms
`has been also described in prokaryotes
`including cyanobacteria,
`Accurately capturing and measuring (quantifying) cell variability and
`PCD in cyanobacterial populations is essential
`for understanding
`cellular and molecular mechanisms
`these ancient organisms.
`of
`In
`flow imaging cytometry
`coupled with combination
`this work, we use
`of nucleic acid and metabolic fluorescent dyes to characterize cell
`variability and PCD in several species of
`filamentous cyanobacteria.
`Aging cyanobacterial cultures were stained with Annexin-V dye,
`used for detection of early stages of apoptosis-like PCD; nucleic acid
`dye Sytox Blue (SB), permeable to damaged and dying cells; and
`Calcein-AM stain that penetrates
`only live cells and is hydrolyzed by
`mtracellular esterases. Control and stained samples were analyzed
`using a 5-laser
`imaging flow cytometer
`Imagestream X Mark
`(Amnis-Millipore
`Inc., Seattle, USA). Different
`cell subpopulations
`mcluding viable cells, apoptotic, and non-viable cells with
`compacted DNA were identified based on Annexin-V, SB staining,
`and morphological
`changes
`chlorophyll
`in DNA structure. Notably,
`autofluorescence was
`in both Annexin-V-positive
`retamed
`and
`SB-positive
`It was also found that co-localization of DNA,
`cells.
`
`II
`
`266
`
`and chlorophyll autofluorescence decreases
`defined
`by SB staining,
`toward the later stages of cell death, whereas
`compactness
`of DNA
`increasing. Calcein-AM staining allowed to differentiate several
`was
`cell subpopulations including vegetative
`cells with chlorophyli
`autofluorescence and varying
`degree of Calcein-AM fluorescence,
`heterocysts with no chlorophyll autofluorescence and either absent
`or very low Calcein-AM signal, and akinetes with strong chlorophyll
`in which Calcein-AM signal was not detected
`autofluorescence,
`at all. Moreover by using algorithms of
`IDEAS software, we were
`and quantitate compaction of DNA in cytoplasm of
`able to
`identify
`undergoing PCD cyanobacteria.
`
`We conclude that multi-color
`imaging flow cytometry
`coupled with
`nucleic acid and metabolic dyes can
`an e×tremely
`advantageous
`be
`approach for studying
`PCD progression and cell variability in
`morphologically
`and functionally diverse cells of
`filamentous
`cyanobacteria.
`
`429/B269
`Evaluation of
`"Su er Bright" Pol mer Dyes
`in 13-
`16-Color Human ImmunophenotypingPanels
`Anna Belkina, Riley Pihl, jennifer Snyder-Cappione
`Flow Cytometry Core Facility,
`Boston University School of
`Medicine, Boston, MA, United States
`Sirigen Group Limited developed unique polymer
`"Brilliant" dyes that
`have becorne a staple of modern multicolor panel design. Polymer-
`
`to
`
`p
`
`a
`
`e
`
`ntly
`
`rou
`launched by eBioscience.
`The performance of
`these new dyes in
`large polychromatic panels is unclear
`to date. Therefore, we tested
`several preparations of
`the Super Bright dyes (such as Super Bright
`two polychromatic fluorescent
`436 and Super Bright 600)
`panels
`in
`(one 13- and one 16-color). Specifically,
`we evaluated
`the spillover
`spread matrices of both panels to evaluate the compatibility of Super
`Bright dyes with other
`fluorochromes in
`a setup with tight placement p
`fluorochrome emissions
`over
`the spectrum. We have also matched
`of
`Super Bright conjugates with comparable BrilliantViolet-labeled
`ahntibodie
`la3olor pahna
`eRr6eand
`ex
`ng
`
`s
`
`C
`
`is
`
`a
`
`×
`
`T
`
`a
`
`se
`
`resting human PßMC cells. Our
`CD25,
`results show that Super
`on
`Bright dyes inflict
`spread in neighboring channels.
`a modest spillover
`16 spillover
`spread matrix
`(3-UV, 5-VIOLET,
`5-BLUE, 3-RED)
`in
`16
`Super Bright dyes demonstrate
`low to moderat
`illover
`that
`ver
`close quantitativel
`the Brilliant Vi
`uman
`to
`immuno
`enotyping pane
`developed to quantify
`we previously
`the "brightness" (i.e.,
`subsets,
`the staining
`index of
`the Super
`cell
`Bright-conjugated antibodies) appears to
`lower than comparable
`be
`Brilliantviolet dyes when titrated,
`u1ations in
`anel are still well
`full
`the use
`arate
`to nine
`of
`As
`up
`nt
`polymer
`dyes simuÍtaneously
`not uncommon, we
`large panels is
`in
`also tested the performance of Super Bright dyes in staining protocols
`include Brilliant Buffer
`that
`to prevent polymer-dye
`(BD Biosciences)
`and found them compatible. O
`interactions
`rform well
`large pol
`anels. This
`Bri
`in
`expansion of commerci
`conjugated a
`ody repertoire
`ava
`with the addition of Super Brights is
`timeÍy and wi!Í greatly facilitate
`the success of
`larger
`(13+ color)
`fluorescent
`panel design.
`
`430/B270
`Acoustophoretic Orientation of Red Blood Cells for
`Diagnosis of Red Cell Health and Pathology
`jordi
`juncà', Michael Ward2,
`Laura G. Rico',
`Bradford2,
`jordi Petriz'
`'Functional Cytornics,
`Josep Carreras Leukaernia
`(l/C), Barcelona, Spain, 2Thermo Fisher
`Research Institute
`Scientific,Eugene,
`OR, USA
`
`jolene
`
`Background: Distortions of
`
`the normal biconcave disc shape for
`
`ISAC 2017 Program and Abstracts
`
`2
`
`