We thank Joshua Marcus for technical support and Jolien Tyler, Ph.D., Director of the Richard J. McIntosh Light Microscopy Core Facility, for technical advice. This work was supported by funds from the University of Colorado Boulder and the University of Colorado Boulder Graduate School to J.D.O. R.T.B. is partially supported by pre-doctoral training grant from the NIH (T32 GM008759). We thank Karyopharm Therapeutics, Inc. for selinexor and Merck Serono for Kinesin-5 inhibitor. FUCCI plasmids are from Atsushi Miyawaki (RIKEN, Japan) via MTA. mCherry-BP1-2 was from Addgene. HeLa expressing H2b-mCherry and &#946;-tubulin-EGFP are from Daniel Gerlich (IMBA, Austrian Academy of Sciences, Austria).

The authors have no conflicts of interest to disclose.

יתרונות של זמן לשגות מיקרוסקופית ו מעקב לאורך שנים המיקרוסקופ הוא מכשיר אידיאלי עבור מחקרים ארוכי טווח התגובה התרופה כפי שהוא מאפשר לחוקרים לעקוב אחר תאים בודדים גורלם וכן את כלל האוכלוסייה. השתנות בתגובת סמים בתוך אוכ...

מיקרוסקופיה Live- תא מעקב אורך של תאים בודדים היא לא טכניקה חדשה. מתוך המיקרוסקופים המוקדמים, חובבי מדענים הבחינו ולומדים תאים בודדים ואורגניזמים, ההתנהגויות שלהם, ופיתוח 1-3. דוגמה מפורסמת מן דיוויד רוג&#39;רס המנוח באוניברסיטת ונדרבילט בשנת 1950 מראה נויטרופילים האדם כתם דם רודפים חיידק סטפילוקוקוס ולבסוף בתהליך של phagocytosis 4. חי בסרט תא זו הוא המחשה מצוינת לאופן שבו מספר רב של תהליכים שניתן לצפות בקורלציה בניסוי יחיד: חישה של שיפוע כימי, מכניקה ומהירות תנועתיות תא, תא מעצבת דינמיקה, הדבקה, ואת phagocytosis של פתוגן. הופעתו של מיקרוסקופים אוטומטיים לחלוטין ומצלמות דיגיטליות רגישות גבוהה הביאה מספר גדל והולך של חוקרים באמצעות מיקרוסקופ לשאול שאלות יסוד f החל הביולוגיה של תאrom כיצד תאים להעביר 5,6 ולחלק 7,8 כדי אברון הדינמיקה וסחר הממברנה 9-11. ללא פלורסנט, מיקרוסקופיה brightfield, כולל שלב בניגוד (PC), אשר זכה בפרס נובל פריץ Zernike בשנת 1953, ואת התערבות ההפרש לעומת זאת (DIC) לאפשר תצפית של תאים גרעינים, אלא גם במבנים המשנה הסלולר כולל חבילות microtubule , כרומוזומים, nucleoli, דינמיקת אברון, וסיבים יקטינו עבים 12. מקודד גנטית חלבוני ניאון לבין התפתחות של צבעי ניאון נגד אברונים השפיעו באופן דרמטי זמן לשגות מיקרוסקופית 13-15. אמנם לא המוקד של מאמר זה, הדמית spheroids תא באתרו (מיקרוסקופיה intravital) באמצעות confocal במיקרוסקופ multiphoton מייצגת הרחבה נוספת של הגישה, ויש מאמרים מצטיינים המשתמשים ולדון הגישות הללו 16-19. התגובות של תאי אל-canc אנטיתרופות אה או מוצרים טבעיים נקבעות על הסולם המולקולרי תאי. לאחר טיפול תגובות גורלות תא הבנה כרוך לעתים קרובות מבחני מיצוע אוכלוסייה (למשל., Immunoblotting, אמצעים היטב שלמים), או בזמן נקודות קבועות עם זיהוי immunofluorescent ו cytometry זרימה, אשר מודדים תאים בודדים. הטרוגניות בתגובות תא בודדות לסמים בקרב אוכלוסייה, בפרט בגידולים, עשויות להסביר חלק ההשתנות בתגובה לראות פני שורות תאים וגידולים כי מטופלים עם התרופה הזהה ב רוויה. לטווח ארוך גישות האורך לעקוב תא בודד נתון או אוכלוסייה של תאים הוא גישה נפוצה פחות, אבל רב עצמה המאפשרת לחקר הישיר של מסלולי תגובה מולקולריים, פנוטיפים שונים (למשל., מוות של תאים או חלוקת תא), תצפית של תא אל תא השתנות בתוך אוכלוסייה, ואיך הגורמים הללו תורמים דינמיקה בתגובת אוכלוסיית 20-22. באופטימיות, יכולתלהתבונן ולכמת התגובות תא בודד יעזור לשפר את הבנתנו כיצד תרופות לעבוד, למה הם לפעמים נכשלים, וכיצד להשתמש בהם בצורה הטובה ביותר. הטכניקה של מיקרוסקופיה לטווח ארוכת זמן לשגות, מעקב אורך, וניתוח תגובות סמים היא העומד לרשות חוקרים רבים והוא יכול להיות פשוט, באמצעות אור המועבר רק להתבונן תגובות פנוטיפ 20,21. הרכיבים העיקריים של הגישה כוללים: ההיערכות מתאימה של התאים בעלי עניין, מיקרוסקופ אוטומטי עם תא סביבתי, מצלמה משולבת עם מחשב לרכוש ולאחסן את התמונות, ותוכנה לעיין לשגות זמן למדוד ולנתח את התאים וכל biosensors ניאון. אנו מספקים פרוטוקול מפורט עם טיפים רבים לניהול מיקרוסקופיה זמן לשגות של תאים בתרבית למשך עוד כמה ימים באמצעות brightfield ו / או מיקרוסקופיה epifluorescent widefield. פרוטוקול זה יכול לשמש לכל שורת תאים שניתן גדלים בתרביתללמוד התגובות שלהם לטיפולים אנטי-סרטניים. אנו מספקים דוגמאות של נתונים רכשה ונותחו באמצעות חיישנים ביולוגיים פלורסנט גנטית בקידוד שונה מרובים דוגמה מיקרוסקופיה שלב בניגוד, לדון סוגים שונים בקצרה של בדיקות, את היתרונות והחסרונות של זמן לשגות לטווח ארוך ומעקב האורך, מה יכול להיות מלומדת הגישה הזאת כי קשה להבין מן הערוצים הלא ישירים, וכמה וריאציות שאנו מקווים יהיה עניין וערך לחוקרים מנוסים שלא נחשבו בגישה, וכדי חוקרים מנוסים.

<table><tbody><tr><td>Taxol (paclitaxel)</td><td>Sigma</td><td>T7191</td><td>microtubule stabilizing drug</td></tr><tr><td>Etoposide</td><td>Selleckchem</td><td>S1225</td><td>topoisomerase II inhibitor</td></tr><tr><td>Selinexor</td><td>Karyopharm Therapeutics</td><td>na</td><td>XPO1/CRM1 inhibitor, gift</td></tr><tr><td>Kinesin-5 inhibitor</td><td>Merck Serono</td><td>na</td><td>gift, also available from American Custom Chemicals Corporation. CAS 858668-07-2</td></tr><tr><td>Cell growth medium</td><td>HyClone (Fisher) or Mediatech</td><td></td><td>many companies available</td></tr><tr><td>5% CO2/balance air, certified</td><td>Airgas</td><td>Z03NI7222004379</td><td></td></tr><tr><td>35 mm Dish, 20 mm glass bottom</td><td>Cellvis</td><td>D35-20-1.5-N</td><td>many companies available</td></tr><tr><td>35 mm 4-well Dish, 20 mm glass bottom</td><td>Cellvis</td><td>D35C4-20-1.5-N</td><td>many companies available</td></tr><tr><td>35 mm Dish, gridded glass bottom</td><td>MatTek</td><td>P35G-2-14-CGRD</td><td>many companies available</td></tr><tr><td>Multi-well, glass bottom</td><td>Cellvis</td><td>P12-1.5H-N</td><td>many companies available</td></tr><tr><td>Olympus IX81 inverted epifluorescence microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Olympus IX2-UCB controller</td><td>Olympus</td><td></td><td></td></tr><tr><td>PRIOR LumenPro200</td><td>Prior Scientific</td><td>Lumen200PRO</td><td></td></tr><tr><td>PRIOR Proscan III motorized stage</td><td>Prio Scientific</td><td>H117</td><td></td></tr><tr><td>STEV chamber</td><td>InVivo Scientific</td><td>STEV.ECU.HC5 STAGE TOP</td><td></td></tr><tr><td>Environmental Controller Unit</td><td>InVivo Scientific</td><td>STEV.ECU.HC5 STAGE TOP</td><td></td></tr><tr><td>Hamamatsu ORCA R2 CCD with controller</td><td>Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>Nikon Eclipse Ti</td><td>Nikon</td><td></td><td></td></tr><tr><td>Nikon laser launch</td><td>Nikon</td><td></td><td></td></tr><tr><td>SOLA light engine</td><td>lumencor</td><td></td><td></td></tr><tr><td>iXon Ultra 897 EM-CCD</td><td>ANDOR&amp;nbsp;</td><td></td><td></td></tr><tr><td>TOKAI HIT inclubation chamber</td><td>TOKAI HIT</td><td>TIZSH</td><td></td></tr></tbody></table>

through the looking glass: time-lapse microscopy and longitudinal tracking of single cells to study anti-cancer therapeutics

התגובה של תאים בודדים לתרופות אנטי-סרטניות תורמת באופן משמעותי בקביעת תגובת האוכלוסייה, ולכן הוא גורם מרכזי תורם בתוצאה הכללית. Immunoblotting, cytometry זרימה וניסויי תא קבוע משמשים לעתים קרובות כדי ללמוד כיצד תאים מגיבים לתרופות אנטי-סרטניות. שיטות אלה חשובות, אבל יש להם חסרונות רבים. משתנה הם תגובות סמים בין סרטן תאים נורמלים, ובין תאים ממוצא הסרטן שונה, וחולף ותגובות נדירות שקשה להבין באמצעות מבחני מיצוע אוכלוסייה וללא יכולת לעקוב ישירות ולנתח אותם אורכים. המיקרוסקופ הוא גם מתאים במיוחד לתאים חיים תמונה. התקדמות הטכנולוגיה מאפשרת לנו תאי תמונה שגרתי ברזולוציה המאפשרת לא מעקב תא בלבד, אלא גם את התצפית של מגוון תגובות הסלולר. אנו מתארים גישה בפירוט המאפשר הדמיה זמן לשגות רציפה שלתאים במהלך תגובת התרופה בעצם כל עוד רצוי, בדרך כלל עד 96 שעות. באמצעות וריאציות של הגישה, ניתן לנטר תאים במשך שבועות. עם העסקה של תהליכים רבים biosensors פלורסנט מקודד גנטי, שבילים ותגובות יכולה להיות אחריו. אנו מציגים דוגמאות הכוללות מעקב וכימות של גדילת התא התקדמות מחזור התא, דינמיקה כרומוזום, נזק לדנ&quot;א, מוות של תאים. כמו כן נדונו וריאציות של הטכניקה והגמישות שלה, ולהאיר כמה מלכודות נפוצות.

הזמן לשגות לטווח ארוך מיקרוסקופיה מעקב אורך ישיר מאפשר לחקר השפעות רבות נגד הסרטן במהלך תגובת תרופה. בעקבות בקווים הכלליים באיור 1, מספר דוגמאות של תאים מוצגות להביע כתבי ניאון תוקפים כי שטופלו בתרופות אנטי-סרטניות, במעקב, ונותחו באמצעות ג?...

Watch this Scientific Journal Video about Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics at JoVE.com

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

מבעד למראה: זמן לשגות מיקרוסקופית ו מעקב אורך של תאים בודדים כדי ללמוד Therapeutics אנטי-סרטניות

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

through-looking-glass-time-lapse-microscopy-longitudinal-tracking

Research

JoVE Journal

Medicine

10.9K Views.  University of Colorado, Boulder. Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Video: Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

לטווח ארוך לחיות תאים הדמיה כדי להעריך את גורל בתגובה פקליטקסל

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Cancer Research

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

האורך הדמיה של התנהגות המוח תאים סרטניים בתגובה ביופסיה פולשנית כירורגית

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

הדמיה הזמן 2D דימות של פעילות Phagocytic ב-M1 מקרופאג-4T1 עכבר קרצינומה של החלב תאים במשותף תרבויות

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

חי הדמיה של תגובות סמים בmicroenvironment הגידול במודלים עכבריים של סרטן השד

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

הדמית זמן לשגות של תאי אפיתל חלב Preneoplastic ראשוניים הנגזרת ממודלי עכבר מהונדס גנטי של סרטן השד

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

זמן לשגות הדמיה של מיטוזה לאחר transfection siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Biology

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

זמן לשגות Extended הדמיה Intravital של Dynamics תאיים בזמן אמת ב microenvironment הגידול

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

חיים הדמיה ואחריו תא בודד מעקב צג Cell Biology, ההתקדמות שושלת היוחסין של אוכלוסיות עצבית מרובים

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Neuroscience

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

שימוש במבחנה לחיות תאים הדמיה כדי לחקור Chemotherapeutics מועברת על ידי חלקיקים המבוססת על שומנים בדם

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

Here, we present a protocol to monitor survival on a single-cell basis and identify variables that significantly predict cell death.

ניטור הישרדות עצביים באמצעות מיקרוסקופ פלורסצנטיות האורך

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to ...