Name: quality-controlled sputum analysis by flow cytometry
Uploaded: 2021-08-09T13:00:00
Description: Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

Vi vil gerne takke David Rodriguez for hans hjælp med figurforberedelsen. Sputum prøver blev kørt på BD LSR II på UT Health San Antonio Flow Cytometry Shared Resource Facility, støttet af UT Health, NIH-NCI P30 CA054174-20 (CTRC på UT Health) og UL1 TR001120 (CTSA tilskud).

Alle trin i spytbehandlingen udføres i et biologisk sikkerhedsskab med passende personlige værnemidler.
1. Reagens forberedelse før start sputum dissociation
<ol><li>Optø 1% Paraformaldehyd (PFA), 25 mL pr. prøve på is, og hold dig kold, indtil den er i brug. FORSIGTIG: PFA er giftig ved indånding og hudkontakt. Fiksativt forberedes i overensstemmelse med fabrikantens anvisninger, og frysning ved -20 °C i 25 mL aliquots indtil brug.</li>
	<li>Prøvens vægt tilnærmes og optøs no...

Det cellulære indhold af spyt omfatter et stort udvalg af vidtrækkende celler, ofte ledsaget af en masse snavs37. Derudover kræver spytanalyse en kvalitetskontrol, der bekræfter, at prøven indsamles fra lungen i stedet for mundhulen38. Derfor er det ikke så simpelt at analysere spyt ved flowcytometri som det er for blod, for eksempel, hvilket frigiver en meget renere og homogen celleaffjedring. Denne protokol har behandlet alle disse spørgsmål: at give instrumentind...

Tekniske fremskridt inden for hardware og software af flowcytometre har gjort det muligt at identificere mange forskellige cellepopulationer samtidigt1,2,3,4. Udnyttelsen af flowcytometeret i hæmatoopoietisk celleforskning har for eksempel ført til en langt bedre forståelse af immunsystemet2 og det cellulære hierarki i det hæmatoopoietiske system5 samt den diagnostiske sondring af en lang række forskellige blodkræft6,7,8. Selv om de fleste spytceller er af hæmatopoietisk oprindelse9,10,11, flow cytometri har ikke været almindeligt anvendt til spyt analyse til diagnostiske formål. Men, flere undersøgelser tyder på, at evalueringen af immuncellepopulationer i spyt (den mest betydningsfulde delmængde af celler) kan være til stor hjælp i diagnosticering og / eller overvågning af sygdomme som astma og kronisk obstruktiv lungesygdom (KOL)12,13,14,15. Desuden tillader eksistensen af epitelspecifikke markører, der kan bruges i flowcytometri, forhør af følgende mest betydningsfulde delmængde af celler i spyt, lungeepileleceller.
Ud over evnen til at analysere mange forskellige cellepopulationer af forskellig vævsoprindelse kan et flowcytometer evaluere et stort antal celler på relativt kort tid. Til sammenligning kræver diasbaserede, cytologiske typer analyser ofte højt specialiseret personale og/eller udstyr. Disse analyser kan være arbejdskrævende, hvilket fører til, at kun en del af spytprøven analyseres16.
Tre kritiske spørgsmål begrænser den udbredte brug af spyt i flowcytometri. Det første nummer vedrører indsamlingen af spyt. Spyt opsamles gennem en huff hoste, der udviser slim fra lungerne ind i mundhulen, efterfølgende spytte ind i en samling kop. Da slim rejser gennem mundhulen, er der en stor chance for SEC forurening. Denne forurening komplicerer prøveanalysen, men problemet afhjælpes let på en flowcytometrisk platform, som vist i denne undersøgelse.
Ikke alle kan producere spyt spontant; derfor er der udviklet flere enheder til at hjælpe med spytsamlingen på en ikke-invasiv måde17. Forstøveren er en sådan anordning og har vist sig at producere pålidelige spytprøver18,19,20. Selv om forstøveren er en meget effektiv måde at ikke-invasivt indsamling spyt, dens anvendelse stadig kræver en indstilling på en medicinsk facilitet med specialiseret personale21. I modsætning hertil kan håndholdte enheder som lungefløjten22, 23,24 og acapella16,25 bruges hjemme, da de er meget brugervenlige. Disse hjælpeenheder er både sikre og omkostningseffektive.
For os gav acapella konsekvent bedre resultater end lungefløjten16, og derfor er acapella-enheden valgt til spytsamlinger. En 3-dages indsamling prøve blev besluttet, fordi det primære formål med at bruge spyt er at udvikle en lungekræft afsløring test16. Det har vist sig, at en 3-dages prøve øger sandsynligheden for lungekræft afsløring i forhold til en 1- eller 2-dages prøve26,27,28. Andre metoder til sputumindsamling kan dog være at foretrække til forskellige formål. Hvis der anvendes en anden spytsamlingsmetode end den, der er beskrevet her, anbefales det omhyggeligt at titrere hvert antistof eller farvestof, der anvendes til flowcytometrianalyse; meget få data er tilgængelige om, hvordan forskellige spyt indsamling metoder påvirker de målrettede proteiner til flow cytometri.
Det andet spørgsmål, der dæmper begejstringen for at bruge spyt til diagnostik, primært relateret til flowcytometri, er cellenummer. Problemet er indsamlingen af tilstrækkelige levedygtige celler til en pålidelig analyse. To undersøgelser viste, at spytprøver indsamlet ved ikke-invasive metoder, ved hjælp af en hjælpeanordning, indeholder nok levedygtige celler, der kan udnyttes i klinisk diagnose eller forskningsundersøgelser16,24. Ingen af disse undersøgelser omhandlede imidlertid spørgsmålet om celletal vedrørende flowcytometri.
For de undersøgelser, der danner grundlag for denne protokol, sputum prøver blev indsamlet fra deltagere med høj risiko for at udvikle lungekræft efter godkendte institutionelle retningslinjer for hvert studiested. Højrisikodeltagere blev defineret som mellem 55-75 år, efter at have røget 30 pakkeår og ikke har holdt op med at ryge inden for de sidste 15 år. Patienterne blev vist, hvordan man bruger acapella-enheden i henhold til producentens anvisninger29 og indsamlede spyt i tre på hinanden følgende dage derhjemme. Prøven blev opbevaret i køleskabet indtil den sidste samling. På den sidste indsamlingsdag blev prøven sendt til laboratoriet natten over med en frossen kold pakke. Prøverne blev behandlet til en enkelt celle suspension den dag, de blev modtaget. Med denne metode til sputumsamling opnås mere end nok levedygtige celler til en pålidelig flowcytometrisk analyse.
Endelig, og relateret til den tidligere celle nummer spørgsmål, er spørgsmålet om, hvordan man frigiver spytceller fra sin mucinøse miljø. Hvordan kan cellerne holdes levedygtige og skabe en enkelt celle suspension, der ikke tilstopper strømmen cytometer? Baseret på indledende arbejde af Pizzichini et al.30 og Miller et al.31, beskriver denne protokol en nem og pålidelig metode til spyt behandling i en enkelt celle suspension, der er egnet til flow cytometrisk analyse. Denne metode har anvendt veletablerede retningslinjer i flowcytometri32,33,34 til at udvikle en effektiv antistofmærkningsstrategi til at identificere hæmatopoietiske og epitelceller i spyt og give instrumentindstillinger, kvalitetskontrolforanstaltninger og analyseretningslinjer, der standardiserer spytanalyse på en flowcytometrisk platform.

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			<td>100 &#181;M nylon cell strainers, Falcon #352360</td>
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quality-controlled sputum analysis by flow cytometry

Sputum, der i vid udstrækning anvendes til at studere det cellulære indhold og andre mikromiljøfunktioner for at forstå lungens sundhed, analyseres traditionelt ved hjælp af cytologibaserede metoder. Dens nytte er begrænset, fordi læsning af dias er tidskrævende og kræver højt specialiseret personale. Desuden gør omfattende snavs og tilstedeværelsen af for mange pladeformede epitelceller (SECs) eller kindceller ofte en prøve utilstrækkelig til diagnose. I modsætning hertil giver flowcytometri mulighed for fænotyping med høj gennemløb af cellulære populationer, samtidig med at snavs og 2-metri'er udelukkes.
Protokollen præsenteres her beskriver en effektiv metode til at adskille spyt i en enkelt celle suspension, antistof plet og fastsætte cellulære populationer, og erhverve prøver på en flow cytometri platform. En gating strategi, der beskriver udelukkelse af vragrester, døde celler (herunder SECs) og celle doublets præsenteres her. Endvidere forklarer dette arbejde også, hvordan man analyserer levedygtige, enkelte spytceller baseret på en klynge af differentiering (CD)45 positive og negative populationer for at karakterisere hæmatoopoietiske og epitelstrenge subsets. En kvalitetskontrol foranstaltning er også fastsat ved at identificere lunge-specifikke makrofager som bevis for, at en prøve er afledt af lungen og er ikke spyt. Endelig er det blevet påvist, at denne metode kan anvendes på forskellige cytometriske platforme ved at levere spytprofiler fra den samme patient analyseret på tre flowcytometre; Navios EX, LSR II og Lyric. Desuden kan denne protokol ændres til at omfatte yderligere cellulære markører af interesse. En metode til at analysere en hel spytprøve på en flowcytometrisk platform præsenteres her, der gør spyt modtagelig for udvikling af høj-throughput diagnostik af lungesygdom.

Denne protokol blev udviklet med et klinisk laboratorium indstilling i tankerne. Fokus under udviklingen af protokollen var på enkelhed, effektivitet og reproducerbarhed. Det blev konstateret, at det mest tidskrævende trin i behandlingen af spyt var at tælle cellerne. Derfor er protokollen konfigureret på en sådan måde, at spytbehandling og cellemærkning kan udføres uafhængigt af celletælling uden tidstab. Et nøjagtigt celletal, som stadig er nødvendigt for at fortynde prøven korrekt til en uhindret kørsel,...

Watch this Scientific Journal Video about Quality-Controlled Sputum Analysis by Flow Cytometry at JoVE.com

Quality-Controlled Sputum Analysis by Flow Cytometry

Denne protokol beskriver en effektiv metode til at adskille spyt i en enkelt celle suspension og den efterfølgende karakterisering af cellulære delmængder på standard flow cytometriske platforme.

Kvalitetsstyret spytanalyse efter flowcytometri

Sputum, widely used to study the cellular content and other microenvironmental features to understand the health of the lung, is traditionally analyzed ...

Denne protokol løser almindelige udfordringer med spytbehandling, der tidligere har gjort det vanskeligt at bruge med flowcytometri til kliniske diagnostiske formål med høj overførselshastighed. Denne protokol er designet til at spare tid ved hjælp af spyt vægt til at bestemme antistof og farve farvning mængder. Under farvning inkubationen kan celletælling udføres for downstream trin.Det er vigtigt at opnå et nøjagtigt celletal for at bestemme den korrekte genaffjedringsvolumen for at forhindre problemer, når du kører på flowcytometeret. Denne protokol kan give indsigt i forskningsområder, der undersøger lungesundhed. For eksempel kan sygdomme som KOL, astma, lungekræft eller virkningerne af COVID undersøges.Demonstrere proceduren vil være Dr.Michael Lai, en forsknings-og udviklingsforsker fra mit laboratorium. Til at begynde med skal du veje spytprøven for at bestemme mængden af dissociationsreagenser. Prøven overføres til det fartøj, der er af passende størrelse, baseret på spytvægten, og tilsæt derefter en milliliter pr. gram på 0,5 % NAC og fire milliliter pr. gram på 0,1 % DTT.Hvirvle prøverne ved maksimal hastighed i 15 sekunder og rock ved maksimal hastighed i 15 minutter ved stuetemperatur. Fortynd denne prøve med 4X Hanks afbalanceret saltopløsning eller HBSS for at neutralisere NAC og DTT. Og efter hurtig hvirvlende, rock prøven ved stuetemperatur i fem minutter.Filter celle suspension gennem en 100 mikrometer nylon mesh celle si i en eller flere 50 milliliter koniske centrifuge rør til at skabe en enkelt celle suspension. Pellet ned cellerne. Efter indsugning af supernatanten kombineres pellets i et 15 milliliter konisk rør og vaskes med HBSS under de samme forhold.Brug cellepillen igen i en buffervolumen bestemt af spytprøvens oprindelige vægt. Tag en aliquot fra celleaffjedringen og bland 10 mikroliter af spytfortyndingen med 30 mikroliter på 0,4% trypanblå, og læg derefter ind i tællekamrene i et hæmocytometer for et levende / dødt celletal. Mærk spyt i erstatningsrør.Tilsæt HBSS antistof og farvestof til hvert spyt celle rør og kompensation rør som angivet i teksten. Tilsæt spytcellerne til analyserørene og tilføj kompensationsperler til kompensationsrørene som nævnt i teksten. Inkuber alle rør på is beskyttet mod lys i 35 minutter.Derefter efter påfyldning af rørene med iskold HBSS, centrifuge ved fire grader Celsius i 10 minutter ved 800 gange G.For kompensation rør, aspirere supernatant så tæt på pellets som muligt og svirp pellets til at løsne. Derefter tilsættes 0,5 milliliter kold HBSS til kompensationsrørene og opbevar rørene på isen i et lysbeskyttet område, indtil det er nødvendigt til flowcytometrianalyse. Derefter aspirere supernatant fra spyt unstained, isotype, blod, og epitel rør.Løsn pillerne ved at svirpe rørene. Tilsæt to milliliter af 1% PFA til de ufarvede og isotyperør og 10 milliliter til blod- og epitelrørene. Inkuber rørene på is på en let beskyttet måde i 30 minutter, hvirvlende hurtigt ved maksimal hastighed og inkuber igen i yderligere 30 minutter.Fyld derefter rørene med iskold HBSS. Dernæst centrifugere rørene ved fire grader Celsius i 10 minutter ved 1,600 gange G.Aspirate så meget supernatant som muligt uden at forstyrre cellepillen og svirp røret med fingrene for at løsne cellerne, derefter tilføje 200 mikroliter af kolde HBSS til unstained og isotype rør. Beregn og tilsæt den nødvendige mængde HBSS til resuspension af blod og epitelrør i henhold til det samlede celletal.Opbevar alle prøverne i erstatningsrør, der er beskyttet mod lys på isen, ved fire grader Celsius, indtil flowcytometrianalysen udføres. Anvend passende startprocedurer for det flowcytometer, der bruges. Brug blandingen af National Institute of Standards and Technology eller NIST perler for at sikre, at fremad scatter og side scatter spændinger er indstillet til at placere NIST perler til at spænde plottet uden at placere perlerne for tæt på akserne.Indstil flowhastigheden til medium for LSRII eller høj for Navios EX. Juster spændingerne for hver parameter, der bruges til scatter- og fluorescerende parametre for at placere cellepopulationerne i stor skala. Brug tallene med gating strategi som vejledning til at justere spændingerne i overensstemmelse hermed. Erhverve data for den ubesmittede spyt prøve først, efterfulgt af isotype farvet prøve, og derefter blodrøret og epitel rør.Sputumvægt blev brugt til at bestemme antistof- eller farvevolumener til farvning. Sammenhængen mellem spytvægt og celleudbytte var ikke stærk. Spytprøver blev opdelt i fire vægtkategorier, som grupperede sig i fordelingen af celleudbytte.Hvert antistof, der blev anvendt, blev titreret i en koncentration på plateaufasen med det højeste farvningsindeks blev valgt som arbejdskoncentration. I alle vægtkategorier var den gennemsnitlige SEC-forurening mindre end 20%, idet procentdelen af levedygtige celler blev vist. Præsenteret er den gating strategi, der anvendes til analyse.NIST perler blev brugt til at indstille en port, som blev anvendt på spyt prøve for at fjerne snavs. Små vragrester blev yderligere elimineret af breddeporten. Levedygtighed farvestof blev brugt til at fjerne døde celler, mens singlet gate fjernet doublets.Anti-CD45 antistofmærkningen gjorde det muligt at udskille levende enkelt spytceller i en blodcelle og et ikke-blodcellerum. Profilerne fra Navios EX, LSRII og Lyric blev sammenlignet. Gating strategi for at fjerne snavs, døde celler, doublets, og sammenligne blod og ikke-blod profiler er vist.Denne teknik gjorde det muligt for os at anvende disse metoder til at opdage lungekræftrisiko til brug som en ikke-invasiv klinisk test.

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High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Flowcytometri cellebaseret assay til påvisning af antistoffer til N-methyl-D-aspartat-receptor eller dopamin-2-receptoren i humant serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Tilpasning af halvautomatiseret Cirkulerende Tumor Cell (CTC) analyser til kliniske og prækliniske forskning applikationer

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Indsamling, Isolation, og flowcytometrisk analyse for Human endocervikale prøver

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Stereologisk og flowcytometri karakterisering af leukocytsubpopulationer i modeller af Forbigående eller permanent cerebral iskæmi

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

Molekylær Probe Optimering konstateres Cell Dødelighed i en Fotosyntetisk Organisme (<em&gt; Microcystis aeruginosa</em&gt;) Under anvendelse af flowcytometri

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

Imaging- og flowcytometri-baserede Analyse af Cell holdning og Cell Cycle i 3D Melanom Sfæroider

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

Metodologi for Sputum induktion og laboratorium behandling

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Karakterisering af immunceller i menneskers fedtvæv ved hjælp af flowcytometri

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

En Flow flowcytometri-baserede analyse til måling af membran potentiale i hjerte Myocytes efter hypoxi/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.