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	<li>Szabo, S. E., Monroe, S. L., Fiorino, S., Bitzan, J., Loper, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+automated+instrument+for+viability+and+concentration+mesurements+of+cryopreserved+hematopoietic+cells.">Evaluation of an automated instrument for viability and concentration mesurements of cryopreserved hematopoietic cells.</a> Lab. Hematol. 10, 109-109 (2004).</li><li>Bonnekoh, B., Wevers, A., Jugert, F., Merk, H., Mahrle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+growth+assay+for+epidermal+cell+cultures+by+their+crystal+violet+binding+capacity.">Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity.</a> Arch. Dermatol. Res. 281, 487-487 (1989).</li><li>Sengupta, N., Rose, S. T., Morgan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Flux+analysis+of+CHO+cell+metabolism+in+the+late+non-growth+phase.">Metabolic Flux analysis of CHO cell metabolism in the late non-growth phase.</a> Biotechnol. Bioeng. 108, 83-83 (2010).</li><li>Zhu, M. M., Goyal, A., Rank, D. L., Gupta, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+elevated+pCO2+and+osmolality+on+growth+of+cells+and+production+of+antibody&#x2013;fusion+protein+B1:+a+case+study.">Effects of elevated pCO2 and osmolality on growth of cells and production of antibody&#x2013;fusion protein B1: a case study.</a> Biotechnol. Prog. 21, (2005).</li><li>Ryan, J. M., Sharf, B. B., Cristofalo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+culture+medium+volume+on+cell+density+and+life-span+of+human+diploid+fibroblasts.">The influence of culture medium volume on cell density and life-span of human diploid fibroblasts.</a> Exp Cell Res. 91, 389-389 (2004).</li><li>Flaherty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tips and Techniques for enhancing your cell culture. , (2009).</li><li>Sanyal, S., Abraham, E. J., Slater, K., Cai, A., Lacey, B., Hartshorn, C., Flaherty, P., McHugh, B., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Scaling up cells in BD Falcon Cell Culture Multi-Flasks. , (2011).</li><li>DiPersio, C. M., Jackson, D. A., Zaret, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+coordinately+modulates+liver+transcription+factors+and+hepatocyte+morphology.">The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.</a> Mol. Cell. Biol. 11, 4405-4405 (1991).</li><li>Grose, R., Hutter, C., Bloch, W., Thorey, I., Watt, F. M., Fassler, R., Brakebusch, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crucial+role+of+&#x03B2;1+integrins+for+keratinocyte+migration+in+vitro+and+during+cutaneous+wound+repair.">A crucial role of &#x03B2;1 integrins for keratinocyte migration in vitro and during cutaneous wound repair.</a> Dev. 129, 2303-2303 (2002).</li></ol>

מחברים כל עובדי בקטון דיקינסון, Biosciences, מגזר יחידת דיסקברי labware.

המחקר הנוכחי מדגים את הגידול בפריון כי העיצוב Flask Multi-מציעה לחוקרים. בעוד חשוב לבצע את השלבים שתוארו לעיל, לקבלת ביצועים מיטביים כאשר באמצעות Multi-צלוחיות, ישנם כמה צעדים קריטיים בפרוטוקול זה נחשבים חיוניים ביותר. אלה כוללות: (i) ערבוב של תאים ריאגנטים באמצעות יציאת לערבב בתוך כלי (ii) העברת Multi-Flask בזווית של 45 מעלות עם כיוון השעון לאחר חלוקת נוזל לתוך כל השכבות (iii) הנחת Flask Multi-שלאחר שטוח לתוך מחיצות החממה. שימוש נכון Flask רב מוביל הייצור של אוכלוסייה הומוגנית תא בתוך כלי אחד כי הוא תרבותי בסביבה כי הוא חופף ל-T-175 צלוחיות (5). כלים אלה מספקים תאים 3 ו - 5 פעמים יותר טביעת דומה כמו בקבוק T-175. כלי 3 ו -5 שכבת מספק 525 ו 875cm 2 של אזור הגידול, בהתאמה ולהציע הן מרחב העבודה שלavings למשתמשים. בתרבית רקמה טיפול Surface ניתן להשוות צלוחיות סטנדרטי ומאפשר מדרוג כלפי מעלה ללא צורך מחדש אופטימיזציה התנאים תרבות קיימים או להתפשר על איכות, ההומוגניות או הביצועים של תאים (6, 7). זה גם מספק עבור השוואה עם נתונים שנאספו בעבר. כלים אלה יכולים להיות מצופים גם עם חומרים כימיים, כגון קולגן, פיברונקטין, poly-D-ליזין לספק תשתית לצמיחה המקצועית המצורף, והבחנה של סוגי תאים מסוימים, כגון 8 hepatocytes, kertainocytes 9, בתאי גזע בוגרים בסרום ללא מדיה ניסוחים. פתרונות ציפוי ניתן להסיר עם שמירה מינימלית נוזל שיורי כדי לצמצם בזבוז של חומרים כימיים בעלי ערך, כמו גם הסרה יעילה של פתרון הציפוי לפני תאים culturing. נפח התקשורת מומלץ אופטימלי לתאי תרבות במגוון רב צלוחיות של 0.142-0.287 מ&quot;ל / 2 ס&quot;מ אשר לתרגם 25-50 מ&quot;ל לכל שכבה. בניגוד בקבוק אחר multilayered, לאהמוצר שלו מציע שילוב יציאת בכלי המאפשר מהירה, כלי ערבוב וכן התפלגות אחידה של תאים ריאגנטים בתוך ובין שכבות של כל כלי. שורות תאים מגוונים, תרבויות יסודי בתאי גזע כבר scaled עד ביעילות באמצעות Multi-צלוחיות. כלים אלה הם יתרון במיוחד ביישומים הדורשים מספר רב של תאים כגון תפוקה גבוהה, סינון, ייצור החיסון, transfections וקטור ויראלי טיפול בתא. חסכון, עבודה הזמן, המרחב ואת ייצור הפסולת מופחתת הן זכיות המפתח של Flask רב לעומת תרבויות קונבנציונלי חד שכבתית כלי. אנחנו יכולים התרבות כ שלוש פעמים את מספר התאים שנקטפו מן 5, T-175 צלוחיות באותו מקום באמצעות 3, 5 רב שכבתיים צלוחיות. יתרון זה של שמירת מקום אינה מוגבלת רק ל-T-175, אך ניתן גם להרחיב את כלי אחר: הרים בקבוק סטנדרטי מנגנון שמתאים לבתים חממות משותף במעבדה 4 ~ רולהימלר בקבוקים (2200 מ&quot;ל) שכל אחד מהם מספק 850cm 2 שטח פנים. באותו אזור, ~ 20, 5 רב שכבתיים צלוחיות ניתן שוכנו ובכך לספק חמש פעמים את פני השטח של צמיחת תאים תרבות. יתר על כן, עם גידול המודעות &quot;לך ירוק&quot;, אפשרויות להפחתת ייצור הפסולת הוא רצוי מאוד. במובן זה, יש 38% (5, T-175 צלוחיות שוקל 640g ~ בעוד אחד, 5 רב שכבתיים Flask שוקל ~ 400 גרם) ירידה ייצור הפסולת באמצעות Multi-צלוחיות לעומת T-175 צלוחיות אלה יתרונות להוביל לירידה בעלויות אחסון פסולת לרשות והתוצאה חיסכון כלכלי למשתמש.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / ציוד </td><td> חברה </td><td> קטלוג # </td><td> תגובות </td></tr><tr><td> 3 שכבת רקמה תרבות שטופלו 525cm 2 </td><td> BD Biosciences </td><td> 353143 </td><td rowspan="21"> <a href="http://www.bdbiosciences.com/cellculture/multiflasks/index.jsp">BD Biosciences תרבית תאים - BD פלקון Multi-צלוחיות</a> </td></tr><tr><td> 5 שכבת רקמה תרבות שטופלו 875cm 2 </td><td> BD Biosciences </td><td> 353144 </td></tr><tr><td> 100 מ&quot;ל פיפטה </td><td> BD Biosciences </td><td> 357600 </td></tr><tr><td> 10 מ&quot;ל פיפטה </td><td> BD Biosciences </td><td> 357551 </td></tr><tr><td> 5 מ&quot;ל aspirating פיפטה </td><td> BD Biosciences </td><td&gt; 357501 </td></tr><tr><td> 50 מ&quot;ל צינור פוליפרופילן חרוטי </td><td> BD Biosciences </td><td> 352070 </td></tr><tr><td> T-175 רקמות בקבוק תרבות שטופלו </td><td> BD Biosciences </td><td> 353028 </td></tr><tr><td> גראם קריסטל ויולט </td><td> BD </td><td> 212525 </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td></tr><tr><td> GMEM </td><td> סיגמא </td><td> G5154 </td></tr><tr><td> RPMI-1640 </td><td> ATCC </td><td> 30-2001 </td></tr><tr><td> מ </td><td> ATCC </td><td> 30-2003 </td></tr><tr><td> טריפסין-EDTA </td><td> Lonza </td><td> CC-5012 </td></tr><tr><td> סרום שור עוברית </td><td> Invitrogen </td><td> 16000-044 </td></tr><tr><td> Tryptose פוספט מרק </td><td> סיגמא </td><td> T8159 </td&gt; </tr><tr><td> BHK-21 תאים </td><td> סיגמא </td><td> 85011433 </td></tr><tr><td> הפ-G2, LnCAP </td><td> ATCC </td><td></td></tr><tr><td> Ecopack 2-293 </td><td> Clontech </td><td></td></tr><tr><td> Paraformaldehyde </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> 15710 </td></tr><tr><td> פוליסטירן חרוז </td><td> PolySciences בע&quot;מ </td><td> 24628-20 </td></tr><tr><td> Bio-Flex צפה Analyzer </td><td> נובה ביו </td><td></td></tr></tbody></table>

scale-up of mammalian cell culture using a new multilayered flask

מספר גדל והולך של יישומים מבוססי תאים דורשים מספר גדול של תאים. שכבה אחת של שימוש T-צלוחיות, כי הם נאותים במהלך התרחבות בקנה מידה קטן, עשוי להיות מסורבל, מייגע זמן רב כאשר מספר גדול של תאים נדרשים. כדי לענות על הצורך הזה, את הביצועים של כלי תרבות חדשה רב שכבתית התא כדי להקל על סולם קל של תאים מן מרובד אחד T-צלוחיות יידונו. צלוחיות שנבדקו זמינים 3 - 5 ו - שכבתיים בפורמט ולאפשר תרבות התאוששות מלאה של שלוש עד חמש פעמים את מספר התאים בהתאמה, בהשוואה ל T-175 צלוחיות. תכונה מרכזית של Flask Multi-BD הוא נמל לערבב / איזון המאפשר מהירה, כלי ערבוב וכן התפלגות אחידה של תאים ריאגנטים בתוך ובין שכבות של כלי כל עקבי לייצר תאים שיכולים להיות מתורבת בסביבה הוא חופף ל-T-175 צלוחיות. העיצוב של אלה Multi-צלוחיות גם מאפשר גגישה פיפטה onvenient להוספת ריאגנטים ותאי ישירות לתוך צלוחיות כמו גם התאוששות יעילה של תאים ריאגנטים ערך מפחיתה את הסיכון של זיהום עקב שוטף. עבור יישומים בהם לשפוך היא המועדפת על pipetting, העיצוב מאפשר שמירה מינימלית נוזל שיורי כדי לצמצם בזבוז של תאים ריאגנטים יקר.

Watch this Scientific Journal Video about Scale-Up of Mammalian Cell Culture using a New Multilayered Flask at JoVE.com

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

תאים לשחק תפקיד מרכזי והגדלת במחקר, ואת גילוי ופיתוח של הרפוי חדש. עם הצורך הזה גובר מספר גדול יותר של תאים אנחנו צריכים דרכים יעילות יותר ויעיל גדל קצירת התאים מצורף תלוי. בקבוק רב שכבתי עם התכונות הנכון יכול לשרת מטרה זו.

Scale-Up של תרבית תאים היונקים באמצעות Flask multilayered חדש

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

scale-up-of-mammalian-cell-culture-using-a-new-multilayered-flask

Research

JoVE Journal

Biology

37.4K Views.  BD Biosciences. תאים לשחק תפקיד מרכזי והגדלת במחקר, ואת גילוי ופיתוח של הרפוי חדש. עם הצורך הזה גובר מספר גדול יותר של תאים אנחנו צריכים דרכים יעילות יותר ויעיל גדל קצירת התאים מצורף תלוי. בקבוק רב שכבתי עם התכונות הנכון יכול לשרת מטרה זו.

Video: Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

לטווח ארוך הדמיה בתאי יונקים באמצעות מיקרוסקופית שדה רחב

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson: Development of New Therapies for Parkinson&#039;s Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

אולה Isacson: פיתוח טיפולים חדשים עבור מחלת פרקינסון

Development of New Therapeutic Applications Using Microfluidics

הפיתוח של יישומים טיפולית חדשה באמצעות מיקרופלואידיקה

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

שיטת לתרבות מוקדם של עוברים צ&#39;יק לשעבר vivo (תרבות חדש)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

ניתוח של עמדת מחזור התא בתאי יונקים

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

קביעת ספירת תאים יונקים, גודל התא ובריאות נייד באמצעות ת Moxi מיני מונה אוטומטי Cell

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

הכנת סרום העכברוש מתאים לתרבות עובר שלמה יונקים

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

משלוח חלבון ישיר לשימוש בתאי יונקי Cys Cell-חדיר<sub&gt; 2</sub&gt; כלי-<sub&gt; 2</sub&gt; Domains אבץ האצבע

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect fat body tissue is usually distributed throughout the insect body. However, it is often concentrated in the abdomen and attached to the abdominal body wall.
The mosquito fat body is the sole source of yolk proteins, which are critical for egg production. Therefore, the in vitro culture of mosquito fat body tissues represents an important system for the study of mosquito physiology, metabolism, and, ultimately, egg production. The fat body culture process begins with the preparation of solutions and reagents, including amino acid stock solutions, Aedes physiological saline salt stock solution (APS), calcium stock solution, and fat body culture medium. The process continues with fat body dissection, followed by an experimental treatment. After treatment, a variety of different analyses can be performed, including RNA sequencing (RNA-Seq), qPCR, Western blots, proteomics, and metabolomics.
In our example experiment, we demonstrate the protocol through the excision and culture of fat bodies from the yellow fever mosquito, Aedes aegypti, a principal vector of arboviruses including dengue, chikungunya, and Zika. RNA from fat bodies cultured under a physiological condition known to upregulate yolk proteins versus the control were subject to RNA-Seq analysis to demonstrate the potential utility of this procedure for investigations of gene expression.

Fat Body Organ Culture System in Aedes Aegypti, a Vector of Zika Virus

The fat body is the central metabolic organ in insects. We present a live organ culture system that enables the user to study the responses of isolated fat body tissue to various stimuli.

תרבות הגוף השמן איברים במערכת Aedes Aegypti, וקטור של וירוס Zika

The insect fat body plays a central role in insect metabolism and nutrient storage, mirroring functions of the liver and fat tissue in vertebrates. Insect ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

צמיחה אנאירובית ותחזוקה של שורות תאים בתרבית

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...