Name: sample preparation for mass cytometry analysis
Uploaded: 2017-04-29T13:00:00.000+00:00
Duration: 6 min 28 s
Description: Watch this Scientific Journal Video about Sample Preparation for Mass Cytometry Analysis at JoVE.com

Work in the Barton lab was supported by a grant from the Cancer Prevention and Research Institute of Texas (CPRIT RP110471). This research was supported in part, by a training grant fellowship for Ryan L. McCarthy from the National Institutes of Health Training Program in Molecular Genetics 5 T32 CA009299. The core facility that maintains and runs the mass cytometry machine is supported by CPRIT grant (RP121010) and NIH core grant (CA016672).

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The authors have nothing to disclose.

Il protocollo presentato qui è stato impiegato con successo per il trattamento di varie linee in coltura di cellule (H1 e H9 hESC, mESCs, MCF7, HEK 293, KBM5, HMEC, MCF10A) e campioni di tessuti primari (midollo osseo di topo, il mouse del fegato embrionale, il mouse per adulti fegato, tumore mouse). Indipendentemente dalla fonte, qualsiasi tessuto che può essere dissociato in singole cellule preservando lo stato cellulare dovrebbe essere suscettibile di analisi mediante citometria di massa, ma può richiedere alcuni ...

Citometria consente la misurazione simultanea di più target anticorpali a livello di singola cellula attraverso grandi popolazioni di cellule. Nel tradizionale citometria di flusso basato sulla fluorescenza, il numero di parametri che possono essere quantificate è limitato dalla sovrapposizione spettrale tra lo spettro di emissione dei fluorofori multipli, che richiede calcoli di compensazione sempre più complessi come il numero di parametri aumenta. Queste limitazioni sono rivolte in massa citometria, dove vengono rilevati e quantificati mediante tempo di volo (TOF) spettrometria di massa per espandere notevolmente il numero di parametri raccolti simultaneamente e produrre un alto dimensionale profilo proteico-abbondanza per ogni singola cella anticorpi metallo coniugati pesanti. Una conoscenza di base del funzionamento della massa citometria strumento è vantaggioso per l&#39;utente e può essere essenziale per la risoluzione dei problemi. Per una descrizione più approfondita di messa a punto e l&#39;esecuzione di una macchina citometria di massa,consultare il relativo manoscritto 1. Brevemente, un campione di cellule è etichettato con un pannello di metallo anticorpi coniugati destinati marcatori di superficie cellulare, le proteine citoplasmatiche, proteine nucleari, proteine cromatina-bound o altri epitopi di interesse (Figura 1i). Le cellule marcate vengono caricate sulla macchina, uno alla volta mediante iniezione manuale o mediante un autocampionatore che i campioni da una piastra a 96 pozzetti. Le cellule caricate vengono iniettate attraverso un nebulizzatore, che genera uno spruzzo di goccioline di liquido che incapsulano le cellule (Figura 1ii). Questo spray è posizionato in modo che le cellule vengono ionizzati da una torcia al plasma di argon. Questo ionizzazione genera una nube di particelle comprendente tutti gli atomi costituenti ciascuna cella (Figura 1iii). Poiché questa nube particella viaggia verso il rivelatore, bassi atomi di massa atomica sono separati dalle alte ioni massa da un filtro di massa a quadrupolo. I restanti alti ioni di massa continuano al rivelatore, dove l&#39;abundance di ciascun isotopo viene quantificato (Figura 1iv). I dati grezzi raccolti dal rivelatore, vengono analizzati dal software dello strumento citometria di massa per identificare gli eventi cellulari. Per ciascun evento cella identificata, il segnale rilevato in ciascun canale viene quantificata e salvata in un file un&#39;uscita .fcs. I canali di massa utilizzati per rilevare gli anticorpi metallo pesante coniugati presentano minima sovrapposizione spettrale, che va da 0,4% con la maggior parte dei contributi inferiori all&#39;1%. A causa di questa cross-talk tra i canali, è generalmente necessario trasformare i dati con una matrice di compensazione prima dell&#39;analisi 2. Tuttavia, occorre prestare attenzione durante la progettazione del pannello sperimentale di anticorpi per garantire che un anticorpo con un epitopo alta abbondanza non è assegnato a un canale di massa che contribuisce segnale ad un canale assegnato ad un anticorpo a bassa abbondanza, in quanto ciò può creare artificialmente elevati della popolazione nel canale di ricezione del segnale del contributo. <p class = &quot;jove_content&quot;&gt; La preparazione esatta di campioni per analisi di citometria di massa dipende fortemente i tipi di campioni che vengono raccolti e l&#39;ipotesi sperimentale in fase di test. Forniamo esempi di due protocolli per la raccolta diversi tipi di cellule (cellule staminali embrionali umane) e cellule primarie (topo tumore mammario isolare cellule epiteliali). Quando si inizia uno studio citometria di massa, si consiglia di effettuare prima un piccolo esperimento pilota per garantire che tutti i protocolli e gli anticorpi per essere utilizzati stanno lavorando bene nelle mani del ricercatore. Questo è particolarmente importante quando devono essere utilizzati anticorpi coniugati personalizzati, come la reazione di riduzione parziale durante la coniugazione anticorpo ha il potenziale di distruggere affinità anticorpale; Pertanto, ogni anticorpo personalizzato deve essere convalidata empiricamente per assicurare l&#39;accuratezza dei dati. Altre considerazioni includono la determinazione se gli anticorpi superficie cellulare da utilizzare nel saggio riconoscerà loro epitopi dopo fissazione o se NEEd da applicare per vivere le cellule. Alcuni fissaggio può impedire il corretto colorazione con anticorpi superficie cellulare come noto verifica con peptide-MHC colorazione 3, 4. Esecuzione di superficie cellulare colorazione anticorpale su cellule vive può essere necessario per alcuni anticorpi, ma esclude la possibilità di codici a barre prima dell&#39;etichettatura anticorpo come maggior parte dei metodi di codici a barre vengono eseguiti dopo fissazione 5 sebbene codici a barre a base di anticorpi 6, 7 è un&#39;eccezione. Nel determinare il numero di cellule da preparare per l&#39;analisi, è importante sapere che solo una frazione delle cellule caricate sulla macchina sarà rilevato e produrre dati. Questa perdita è dovuta in primo luogo a inefficienze quando il nebulizzatore spray campione al cannello. La perdita percentuale esatta dipende dalla massa citometria configurazione della macchina e tipo cellulare, ma può variare da 50-85% e deve essereconsiderati quando si progetta l&#39;esperimento. Questo protocollo è stato ottimizzato per 2x10 6 cellule per campione, ma può essere adatta a lavorare campioni di dimensioni più piccole o più grandi. Questo protocollo può essere utilizzato con minime modifiche per dimensioni del campione da 1 x 10 6 a 4 x 10 6, tuttavia è fondamentale che la dimensione del campione essere mantenuta costante in un esperimento. Cambiamenti nel numero di cellule possono influenzare l&#39;intensità dei codici a barre e colorazione degli anticorpi ed essere comunque ridotti all&#39;incirca costante attraverso campioni da confrontare direttamente. Alcune procedure, come l&#39;etichettatura di cellule morte e l&#39;etichettatura del DNA, devono essere effettuate nella stragrande maggioranza, se non tutti i disegni sperimentali. L&#39;etichettatura di cellule morte in esperimenti analizzare solo marcatori di superficie può essere ottenuta utilizzando Rh103, ma Rh103 dovrebbe essere evitato per esperimenti in cui è richiesta permeabilizzazione come si verifica una perdita di colorazione. Per gli esperimenti che coinvolgono permeabilizzazione, si consiglia di utilizzare cisplatino Campioni codici a barre possono ridurre il consumo di anticorpi, ridurre il tempo di acquisizione ed eliminare campione a campione variabilità 9. Il processo di barcoding comporta l&#39;applicazione di un codice specifico di esempio unico per tutte le celle, che viene utilizzato per assegnare ogni cella per il campione di origine. Dopo barcoding i campioni possono essere riuniti prima della colorazione anticorpale, un processo che assicura tutti i campioni sono macchiati ugualmente. Per ridurre al minimo errore sperimentale, è prudente codice a barre e piscina i campioni che devono essere direttamente confrontate tra di loro. Attualmente esistono diversi metodi per codici a barre dei campioni per l&#39;analisi di citometria di massa 5, 6, 7, due dei quali sono qui presentati (vedi sotto). Con Reagen attualmente disponibilits è possibile quantificare l&#39;abbondanza di 38 bersagli degli anticorpi, identificare le cellule in fase S 10, distinguere le cellule vive e morte 8 e misurare i livelli cellulari di ipossia 11 in un esperimento multiplex di campioni multipli. Questa capacità di raccogliere dati monocellulari alta parametro per grandi popolazioni cellulari consente una migliore profilatura di popolazioni cellulari altamente eterogenee e ha già prodotto una serie di dati biologici nuovi 12, 13, 14, 15, 16. Distillando i dati complesso risultante per informazioni interpretabili ha richiesto l&#39;uso di algoritmi di calcolo includendo Spanning Tree progressione della densità Eventi normalizzati (SPADE) 17 e 18 viSNE. Questo articolo presenta una accessibilipanoramica di come progettare e realizzare una massa citometria esperimento e introduce analisi di base della massa citometria dati.

<table><tbody><tr><td>mTeSR1 medium kit</td><td>Stem Cell technologies</td><td>05850</td><td>Warm at room temperature before use</td></tr><tr><td>DMEM F-12</td><td>ThermoFisher</td><td>11330-032</td><td>Warm at 37&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Accutase</td><td>Stem Cell technologies</td><td>07920</td><td>Warm at 37&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Bovine serum albumin</td><td>Equitech</td><td>BAH62</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Cell ID Pt194</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cell ID Pt195</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cell ID Pt196</td><td>Fluidigm</td><td></td><td>Provided at 1&amp;nbsp;mM</td></tr><tr><td>Cisplatin</td><td>Enzo Life Sciences</td><td>ALX-400-040-M250</td><td>Soluble to 25&amp;nbsp;mg/mL in DMSO</td></tr><tr><td>Cell-ID 20-plex Pd Barcoding Kit</td><td>Fluidigm</td><td>201060</td><td></td></tr><tr><td>5-Iodo-2&#039;-deoxyuridine</td><td>Sigma-Aldrich</td><td>I7125</td><td>Soluble to 74&amp;nbsp;mg/mL in 0.2&amp;nbsp;N NaOH</td></tr><tr><td>Rhodium 103 intercelating agent</td><td>Fluidigm</td><td>201103A</td><td></td></tr><tr><td>Methanol</td><td>Fisher</td><td>BP1105-4</td><td>Chill at -20&amp;nbsp;&amp;deg;C before use</td></tr><tr><td>Sodium Azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>5 mL round bottom tubes</td><td>Falcon</td><td>352058</td><td></td></tr><tr><td>5 mL 35 &amp;#956;m filter cap tubes</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>EQ four element calibration beads</td><td>Fluidigm</td><td>201078</td><td></td></tr><tr><td>Hyaluronidase Type I-S</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>Collagenase from Clostridium histolyticum</td><td>Sigma-Aldrich</td><td>C9891</td><td></td></tr><tr><td>Disposable Scalpel #10</td><td>Sigma-Aldrich</td><td>Z69239</td><td></td></tr></tbody></table>

sample preparation for mass cytometry analysis

Massa utilizza citometria anticorpi coniugati con le etichette di metalli pesanti, un approccio che ha notevolmente aumentato il numero di parametri e opportunità per l&#39;analisi pozzo profondo di quanto sia possibile con i tradizionali flusso basato sulla fluorescenza citometria. Come per ogni nuova tecnologia, ci sono passaggi critici che aiutano a garantire la generazione affidabile di dati di alta qualità. Qui presentata è un protocollo ottimizzato che comprende molteplici tecniche per il trattamento di campioni di cellule per l&#39;analisi di citometria di massa. I metodi descritti qui aiuterà l&#39;utente a evitare problemi comuni e raggiungere risultati coerenti minimizzando variabilità, che può portare a dati accurati. Per informare disegno sperimentale, la logica alla base operazioni opzionali o alternative nel protocollo e la loro efficacia nello scoprire nuove scoperte nella biologia del sistema oggetto di indagine è coperto. Infine, i dati rappresentativi è presentato per illustrare i risultati attesi dalle tecniche Presented qui.

Il protocollo presentato qui può essere ampiamente applicata ad una vasta gamma di campioni di cellule in coltura e primario con modifiche solo minori. A seconda delle esigenze dell&#39;esperimento, può essere eseguita in modo modulare quando alcuni elementi, come codici a barre o superficie cellulare colorazione, non sono necessarie. Utilizzato nella sua interezza questo protocollo consente la preparazione di massa multiplato citometria campioni etichettati per identificazione popolaz...

Watch this Scientific Journal Video about Sample Preparation for Mass Cytometry Analysis at JoVE.com

Sample Preparation for Mass Cytometry Analysis

This article describes the collection and processing of samples for mass cytometry analysis.

Preparazione del campione per l&#39;analisi di massa Citometria

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

sample-preparation-for-mass-cytometry-analysis

Research

JoVE Journal

Biology

16.0K Views.  The University of Texas MD Anderson Cancer Center. This article describes the collection and processing of samples for mass cytometry analysis.

Video: Sample Preparation for Mass Cytometry Analysis

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Massa Citometria: Protocollo per l&#39;ottimizzazione giornaliera e campioni di cellule in esecuzione su un citometro Messa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Ricerca

Bioingegneria

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

Isolamento e colorazione dei Keratinociti della pelle del topo per l'analisi specifica del ciclo cellulare dell'espressione proteica cellulare da citometria di massa

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Biologia dello sviluppo

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.

Raccolta, isolamento e analisi citofluorimetrica di campioni endocervicali umano

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

Medicina

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Identificazione e isolamento di progenitori eritroidi dell'unità burst-forming e dell'unità formante colonie da tessuto murino mediante citometria a flusso

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cella singola analisi di Immunophenotype e produzione di citochine nel sangue periferico intero tramite citometria a massa

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...

Immunologia e infezioni

Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a myeloid-biased 39-antibody CyTOF panel to evaluate the hematopoietic system with a focus on the neutrophil-lineage cells by using this protocol. The CyTOF result was analyzed with an open-resource dimensional reduction algorithm, viSNE, and the data was presented to demonstrate the outcome of this protocol. We have discovered new neutrophil-lineage cell populations based on this protocol. This protocol of fresh whole BM preparation may be used for 1), CyTOF analysis to discover unidentified cell populations from whole BM, 2), investigating whole BM defects for patients with blood disorders such as leukemia, 3), assisting optimization of fluorescence-activated flow cytometry protocols that utilize fresh whole BM.

Here, we present a protocol to process fresh bone marrow (BM) isolated from mouse or human for high-dimensional mass cytometry (Cytometry by Time-Of-Flight, CyTOF) analysis of neutrophil-lineage cells.

Preparazione del midollo osseo intero per l'analisi della citometria di massa delle cellule di lignaggio neutrofilo

In this article, we present a protocol that is optimized to preserve neutrophil-lineage cells in fresh BM for whole BM CyTOF analysis. We utilized a ...

Studi metabolici a basso input nei tipi di cellule di topo

Targeted Metabolomics on Rare Primary Cells

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

Autore in primo piano: Progressi nell'analisi metabolomica delle cellule staminali ematopoietiche rare 

Here, we present a protocol to accurately and reliably measure metabolites in rare cell types. Technical improvements, including a modified sheath fluid for cell sorting and the generation of relevant blank samples, enable a comprehensive quantification of metabolites with an input of only 5000 cells per sample.

Metabolomica mirata su cellule primarie rare

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule ...

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and extremely large cell (40-60 &#181;m). As opposed to the fast-increasing knowledge in megakaryopoiesis at the cell biology and molecular level, the characterization of megakaryopoiesis by flow cytometry is limited to the identification of mature MKs using lineage-specific surface markers, while earlier MK differentiation stages remain unexplored. Here, we present an immunophenotyping strategy that allows the identification of successive MK differentiation stages, with increasing ploidy status, in human primary sources or in vitro cultures with a panel integrating MK specific and non-specific surface markers. Despite its size and fragility, MKs can be immunophenotyped using the above-mentioned panel and enriched by fluorescence-activated cell sorting under specific conditions of pressure and nozzle diameter. This approach facilitates multi-Omics studies, with the aim to better understand the complexity of megakaryopoiesis and platelet production in humans. A better characterization of megakaryopoiesis may pose fundamental in the diagnosis or prognosis of lineage-related pathologies and malignancy.

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Here, we present an immunophenotyping strategy for the characterization of megakaryocyte differentiation, and show how that strategy allows the sorting of megakaryocytes at different stages with a fluorescence-activated cell sorter. The methodology can be applied to human primary tissues, but also to megakaryocytes generated in culture in vitro.

Preparazione del campione per l'analisi di massa Citometria

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Capitoli in questo video

Massa Citometria: Protocollo per l'ottimizzazione giornaliera e campioni di cellule in esecuzione su un citometro Messa CyTOF

Isolamento e colorazione dei Keratinociti della pelle del topo per l'analisi specifica del ciclo cellulare dell'espressione proteica cellulare da citometria di massa

Raccolta, isolamento e analisi citofluorimetrica di campioni endocervicali umano

Identificazione e isolamento di progenitori eritroidi dell'unità burst-forming e dell'unità formante colonie da tessuto murino mediante citometria a flusso

Cella singola analisi di Immunophenotype e produzione di citochine nel sangue periferico intero tramite citometria a massa

Preparazione del midollo osseo intero per l'analisi della citometria di massa delle cellule di lignaggio neutrofilo

Metabolomica mirata su cellule primarie rare

Metabolomica mirata su cellule primarie rare

Metabolomica mirata su cellule primarie rare

Immunofenotipizzazione e smistamento cellulare di MK umani da fonti primarie umane o differenziati in vitro da progenitori ematopoietici