פרויקט זה מומן בחלקו על ידי כספי ממדינת פלורידה סקריפס ומספרי הפרס R01GM085079 ו R21HD061304 מהמכון הלאומי למדעי הרפואה הכללית לבין המכון הלאומי לבריאות הילד והתפתחות האדם, בהתאמה. זהו כתב היד מספר 20917 של מכון המחקר סקריפס.

פרוטוקול זה ניתן להפריד בין שני שלבים עיקריים: (1) ניתוק ו Hoechst 33342 מכתים של תאים עכבר testis ואחריו, במידת הצורך, (2) FACS מיון של שברים meiotic הרלוונטיים, כולל כל שלבי המיוזה, מתאי נבט אל בסיבוב spermatids. לאחר שנאספו, אוכלוסיות אלה meiotic מועשר יכול לשמש עבור מגוון רחב של ניתוח. פרוטוקול זה מתאר את ניתוק של אחד testis מבוגר; כרכים ניתן להתאים בהתאם לנערים או האשכים נוספים. 1. Testis דיסוציאציה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מקום אחד testis decapsulated בצינור 15 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הוסף 3 מ&quot;ל של הפתרון של גיי מאזן מלח (GBSS) המכיל 120 U / ml של collagenase מסוג I. </li><li style=";text-align:right;direction:rtl"> הוסף 10 μl של DNAse אני (פתרון 1mg/ml המניות גליצרול 50%) ולנער אותו נמרצות ביד עד שתראה tubules האשכים מתחילים לנתק. </li><li style=";text-align:right;direction:rtl"> להתסיס אופקית מקסימלית של 120 סל&quot;ד במשך 15 דקות על 33 ° C. </li><li style=";text-align:right;direction:rtl"> למזוג דקות 1 אנכית בטמפרטורת החדר וזורקים supernatant. </li><li style=";text-align:right;direction:rtl"> חזור על שלבים 1.2-1.5. </li><li style=";text-align:right;direction:rtl"> הוסף 2.5 מ&quot;ל של GBSS המכיל סוג collagenase אני, 50 μl של פתרון טריפסין 50mg/ml מניות resuspended בתמיסת HCl 1 מ&quot;מ, ו 10 μl של DNAse אני (1mg/ml), וכן להפוך את הצינור מספר פעמים. </li><li style=";text-align:right;direction:rtl"> להתסיס אופקית מקסימלית של 120 סל&quot;ד במשך 15 דקות על 33 ° C. </li><li style=";text-align:right;direction:rtl"> שימוש pipet פלסטיק חד פעמיות פסטר עם פתח רחב, פיפטה בעדינות מעלה ומטה במשך 3 גושים min.No צריך להיות גלוי בשלב זה. </li><li style=";text-align:right;direction:rtl"> הוסף 30 μl של טריפסין, 10 μl של DNAse אני, 40 μl של Hoechst 33342 resuspended ב DMSO (10 מ&quot;ג / מ&quot;ל), וגם להפוך את הצינור מספר פעמים. </li><li style=";text-align:right;direction:rtl"> להתסיס אופקית מקסימלית של 120 סל&quot;ד במשך 15 דקות על 33 ° C. </li><li style=";text-align:right;direction:rtl"> הוסף 400 μl של עוברי עגלים בסרום (FCS) ומערבבים על ידי היפוך להשבית טריפסין. </li><li style=";text-align:right;direction:rtl"> מכתים הסופי מתבצע על ידי הוספת 50 μl של Hoechst 33342 resuspended ב DMSO (10 מ&quot;ג / מ&quot;ל), וכן 10 μl של DNAse אני (1 מ&quot;ג / מ&quot;ל). </li><li style=";text-align:right;direction:rtl"> להתסיס אופקית מקסימלית של 120 סל&quot;ד במשך 15 דקות על 33 ° C. </li><li style=";text-align:right;direction:rtl"> המדגם testis ניתק לאחר מכן עבר דרך שני GBSS 40 מיקרומטר מראש הרטובות מסננים חד פעמיות על שפופרת 50 מ&quot;ל חרוטי, ולאחר מכן 5 μl של יודיד propidium (PI) פתרון נוסף מדגם מעורבת pipetting בעדינות על ידי מספר פעמים עם פסטר הפנויה פיפטה. </li><li style=";text-align:right;direction:rtl"> לדוגמה מועבר מזרק 5 מ&quot;ל פלסטיק באמצעות מחט 18-מד. האחרון הוא הוחלף על ידי מחט 22-מד למסירה המדגם. מזרק מאוחסן על קרח מוגן מפני האור עד מוכן לעיבוד FACS. </li></ol> 2. FACS התקנה טיהור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מיון בוצעה על סדרן בקטון דיקינסון, תא IIu Aria. התנאים המתוארים ולכן צריך לשמש כנקודת מוצא במיוחד בעת השימוש במכשירים חשמליים שונים. השתמשנו בתנאים שתוארו קודם לכן 7,8. </li><li style=";text-align:right;direction:rtl"> מאז IIu Aria אין לייזר UV, התאמנו את פרוטוקול לאיתור מכתים Hoescht באמצעות לייזר סגול 405nm בנוסף השתמשנו לייזר כחול 488nm לגילוי פיזור קדימה בצד. יתר על כן, מסננים כמה שונו על מנת להגביל כמה leakiness לייזר אדום וכתוצאה מכך חדות משופרת העלילה מפוזרים. </li><li style=";text-align:right;direction:rtl"> לייזר סגול הוגדר לעבור עם להקת 450/40nm לגילוי Hoescht כחול הפליטה לעבור הלהקה 585/42nm עבור פליטת האדום Hoescht. עובר זמן רב 502nm שימש נפרד כחול פלואורסצנטי אדום. </li><li style=";text-align:right;direction:rtl"> זרבובית 100 מיקרומטר היה בשימוש עם תדר טיפה הכונן של 28,000 טיפות / שנייה. שיעור סף המדגם היה כ 4000 אירועים / שנייה. אפשרות בקרת טמפרטורה שימש כדי לשמור על מדגם וצינורות לאיסוף 4 ° C משך כל מיון. בנוסף, תכונת תסיסה מדגם שימש בסל&quot;ד 200 כדי למנוע את הדוגמה sedimenting ברחבי כזה. </li><li style=";text-align:right;direction:rtl"> בדרך כלל, 2-3 האשכים עובדו לכל סוג 6 שעות המאפשר את איסוף 0.5-2.0x10 6 תאים לכל אוכלוסיית (ראה סעיף נציג). </li><li style=";text-align:right;direction:rtl"> המדגם מיון aliquots של כ 750μl להימכר המזרק. בינתיים, במהלך הפסקות אלה הצינורות אוסף הוחזקו על 4 ° C, מוגן מפני האור, מעורב בעדינות לפני מעין חידוש. </li><li style=";text-align:right;direction:rtl"> הדגימות נאספו מיון לתוך צינור איסוף 12x75mm borosilicated זכוכית המכיל 250μl DMEM FCS בתוספת 10%. </li></ol> 3. נציג תוצאות: פרופיל טיפוסי FACS מוצג באיור 1. כל השלבים העיקריים של המיוזה מזוהים מצוינים במקרא. אם Hoechst 33342 הכתם אינו אופטימלי, פרופיל העולמית יופיעו הרבה יותר קומפקטי פחות מוגדר. בעיה נפוצה לגבי הוספת DNAse לא מספיק, אשר יניעו clumping תאים מהירה. תוספת נוספת DNAse בכל הצעדים נגדו כתב אישום בדרך כלל פותר בעיה זו. הקפאת הawing של המניות DNAse (המאוחסן ב -20 ° C) צריכה להיות מינימלית, כמו זו גורמת לאובדן מהיר של פעילות האנזים. Spo11 הוא endonuclease meiotic שמנתב פעמיים גדיל הפסקות באתרים של נקודות חמות רקומבינציה meiotic 12. איור 2 מציג פרופיל אופייני של Spo11-null testis שבו גדיל כפול לשבור לא נוצרים, וכתוצאה מכך המיוזה הנפל 12, כמו גם טרום puberous פרופיל של עכבר 13 ימים הישן, אשר חושף הטבע אסינכרוני של הגל הראשון של המיוזה. תקן מנתח cytogenetic באמצעות שילוב של חלבון מורכב synaptonemal 3 (SCP3) ו phosphorylated נוגדנים H2AX היסטון הבמה הסמנים הספציפיים המיוזה יש להשתמש תחילה אישר את הטוהר ואת אופיו של התאים meiotic מיון, כמתואר elswhere 11. <img src="/files/ftp_upload/2602/2602fig1.jpg" alt="איור 1" /> באיור 1. Wild-type FACS פרופילים המיוזה. א) פיזור העלילה נציג מוצג. התאים מגודרת מוצגים. שים לב כמות גדולה של פסולת נוכח testis מבוגר המכילים בעיקר ממברנות ריקים זנבות spermatid. ב) עלילה נציג לצרכן מראה את כמות מוגבלת מאוד של תאים לצרכן חיובי בהווה במדגם. שער טיפוסי מוצג. ג) נציג wild-type Hoechst 33342 פרופיל FACS מוצג. האוכלוסיות השונות תא meiotic שניתן מטוהרים בשיטה זו למחקר נוסף מצוינים: spermatogonia (SP), טרום leptotene (PL), leptotene-zygotene (L / Z), מוקדם Pachytene (EP), הבינוני Pachytene ( MP), בשלהי Pachytene (LP), diplotene (ד &#39;), סביב spermatids (RS). <img src="/files/ftp_upload/2602/2602fig2.jpg" alt="איור 2" /> איור 2. Spo11-null ו wild-type 13 יום פרופילים FACS meiotic. א) פרופיל טיפוסי Spo11-null מוצג עם כישלון meiotic ברור, שם לא בשלב בעבר leptotene / zygotene מוקדם pachytene ניתן לאתרם. ב) פרופיל של עכבר wild-type 13 יום בן זכר מראה את ריכוז גבוה של תאים leptotene / zygotene. זהו הגל הראשון של המיוזה היא סינכרונית למדי, בבהירות דמיינו שימוש במתודולוגיה זו.

<ol>
	<li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Grabske, R. J., Lake, S., Gledhill, B. L., Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugal+elutriation:+separation+of+spermatogenic+cells+on+the+basis+of+sedimentation+velocity.">Centrifugal elutriation: separation of spermatogenic cells on the basis of sedimentation velocity.</a> J. Cell. Physiol. 86, 177-189 (1975).</li><li>Purification, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=culture,+and+fractionation+of+spermatogenic+cells.">culture, and fractionation of spermatogenic cells.</a> Methods Enzymol. 225, 84-113 (1993).</li><li>Romrell, L. J., Bellve, A. R., Fawcett, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity.+A+morphological+characterization.">Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.</a> Dev. Biol. 49, 119-131 (1976).</li><li>Meistrich, M. L., Trostle, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+mouse+testis+cells+by+equilibrium+density+centrifugation+in+renografin+gradients.">Separation of mouse testis cells by equilibrium density centrifugation in renografin gradients.</a> Exp. Cell Res. 92, 231-244 (1975).</li><li>Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmeiotic+sex+chromatin+in+the+male+germline+of+mice.">Postmeiotic sex chromatin in the male germline of mice.</a> Curr. Biol. 16, 660-667 (2006).</li><li>Lassalle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+Population'+cells+in+adult+mouse+testis+express+Bcrp1+gene+and+are+enriched+in+spermatogonia+and+germinal+stem+cells.">Side Population' cells in adult mouse testis express Bcrp1 gene and are enriched in spermatogonia and germinal stem cells.</a> Development. 131, 479-487 (2004).</li><li>Bastos, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+characterization+of+viable+meiotic+and+postmeiotic+cells+by+Hoechst+33342+in+mouse+spermatogenesis.">Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.</a> Cytometry A. 65, 40-49 (2005).</li><li>Chowdhury, R., Bois, P. R., Feingold, E., Sherman, S. L., Cheung, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+variation+in+human+meiotic+recombination.">Genetic analysis of variation in human meiotic recombination.</a> PLoS Genet. 5, e1000648-e1000648 (2009).</li><li>Roig, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+TRIP13/PCH2+is+required+for+recombination+and+normal+higher-order+chromosome+structure+during+meiosis.">Mouse TRIP13/PCH2 is required for recombination and normal higher-order chromosome structure during meiosis.</a> PLoS Genet. 6, (2010).</li><li>Getun, I. V., Wu, Z. K., Khalil, A. M., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleosome+occupancy+landscape+and+dynamics+at+mouse+recombination+hotspots.">Nucleosome occupancy landscape and dynamics at mouse recombination hotspots.</a> EMBO Rep. 11, 555-560 (2010).</li><li>Romanienko, P. J., Camerini-Otero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+Spo11+gene+is+required+for+meiotic+chromosome+synapsis.">The mouse Spo11 gene is required for meiotic chromosome synapsis.</a> Mol. Cell. 6, 975-987 (2000).</li></ol>

אין ניגודי אינטרסים הכריז.

פרוטוקול המוצג לעיל מאפשר בו זמנית לטהר מעכברים זכר בוגר את כל טווח של תאים בשלב meiotic עם הטוהר גבוהה מאוד, ומאפשר לחוקרים ללמוד את הדינמיקה של התהליך הבסיסי הזה. תאים מטוהרים יכול לשמש עבור יישומים רבים, החל 9,10 מיצוי RNA, מיפוי הנוקלאוזום 11, גילוי מולקולה רקו...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Collagenase Type-1</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>CLSS1</td>
<td>Dissolve to 12,000U/ml in GBSS, store at 4&deg;C</td>
</tr>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>TRL3</td>
<td>Dissolve in 1mM HCl to 50mg/ml, store at 4&deg;C</td>
</tr>
<tr>
<td>Hoechst 33342</td>
<td>&nbsp;</td>
<td>Arcos</td>
<td>230001000</td>
<td>Saturated solution (10mg/ml in DMSO, store at 4&deg;C</td>
</tr>
<tr>
<td>DNAse I</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>DNEP</td>
<td>Dissolve in water/50% glycerol to 1mg/ml, store at -20&deg;C</td>
</tr>
<tr>
<td>Gey&#x2019;s Balance Salt Solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G9779</td>
<td>&nbsp;</td>
<tr>
<td>6" Transfer Pipet</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>137119D</td>
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flow cytometry purification of mouse meiotic cells

אופי הטרוגני של סוגי תאים ב testis ואת היעדרם של מודלים תרבות meiotic תא היו מכשולים משמעותיים כדי ללמוד את התוכניות התמיינות ייחודית כי הם לידי ביטוי במהלך המיוזה. שתי השיטות העיקריות פותחו כדי לטהר, בדרגות שונות, שברים meiotic שונים למבוגרים והן בבעלי חיים מפותחים: elutriation או Staput (שקיעה) באמצעות BSA ו / או percoll הדרגתיים. שתי שיטות אלה מסתמכים על גודל התא וצפיפות להפריד תאים meiotic 1-5. בסך הכל, למעט אוכלוסיות תאים ספורים 6, פרוטוקולים אלה אינם מצליחים להניב טוהר מספקת של אוכלוסיות רבות תא meiotic הדרושים ניתוחים מולקולריים מפורט. יתר על כן, עם שיטות כאלה בדרך כלל סוג אחד של תאים meiotic יכול להיות מטוהרים בזמן נתון, אשר מוסיפה רמה נוספת של מורכבות לגבי שחזור והומוגניות כאשר משווים דגימות תאים meiotic. כאן אנו מתארים שיטה מעודן המאפשר אחד כדי לחזות בקלות, לזהות ולטהר תאים meiotic, החל בתאי הנבט כדי spermatids עגול, באמצעות FACS בשילוב עם מכתים 33342 Hoechst 7,8. שיטה זו מספקת תמונה כוללת של תהליך meiotic כולו מאפשר מאוד לטהר את התאים קיימא משלב ביותר של המיוזה. תאים אלה מטוהרים אז יכול להיות ניתח בפירוט את השינויים המולקולריים שמלווים התקדמות דרך המיוזה, שינויים בביטוי הגנים למשל 9,10 ואת הדינמיקה של תפוסה הנוקלאוזום על נקודות חמות של רקומבינציה meiotic 11.

Watch this Scientific Journal Video about Flow Cytometry Purification of Mouse Meiotic Cells at JoVE.com

Flow Cytometry Purification of Mouse Meiotic Cells

שיטה יעילה להשגת מטוהרים שברים meiotic קיימא מהעכבר testis מתואר, המשלבת תא מעודן דיסוציאציה פרוטוקול עם מיון פלורסנט התא הפעיל (FACS). שיטה זו מנצלת את ההבדלים בתוכן דנ&quot;א צפיפות גרעינית של שברים meiotic בדידים.

Cytometry זרימה טיהור תאים עכבר meiotic

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

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Unit 1

Meiosis

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17.5K Views.  The Scripps Research Institute. שיטה יעילה להשגת מטוהרים שברים meiotic קיימא מהעכבר testis מתואר, המשלבת תא מעודן דיסוציאציה פרוטוקול עם מיון פלורסנט התא הפעיל (FACS). שיטה זו מנצלת את ההבדלים בתוכן דנ"א צפיפות גרעינית של שברים meiotic בדידים.

Video: Flow Cytometry Purification of Mouse Meiotic Cells

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

הכתמה של חלבונים עם ג&#39;ל Coomassie G-250 ללא חומצה אצטית ממס אורגני

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

טיהור הזרימה Cytometry מבוסס<em&gt; ס cerevisiae</em&gt; זיגוטות

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

אופטימיזציה שיטות דוגמנות מכתימה והפצת נשק לניטור חלוקת תא באמצעות צבעי מעקב סלולרי

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

מדידות סידן cytosolic בתאי האפיתל כליות על ידי cytometry הזרימה

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic ציר ההערכה בעכבר ביציות על ידי השתקה בתיווך siRNA

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

LED Thermo Flow &#8212; Combining Optogenetics with Flow Cytometry

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the controlled delivery of light to the cell sample and hence the most popular tools for optogenetic studies are microscopy-based cell analyses and in vitro experiments. The flow cytometer has major advantages over a microscope, including the ability to rapidly measure thousands of cells at single cell resolution. However, it is not yet widely used in optogenetics. Here, we present a device that combines the power of optogenetics and flow cytometry: the LED Thermo Flow. This device illuminates cells at specific wavelengths, light intensities and temperatures during flow cytometric measurements. It can be built at low cost and be used with most common flow cytometers. To demonstrate its utility, we characterized the photoswitching kinetics of Dronpa proteins in vivo and in real time. This protocol can be adapted to almost all optically controlled substances and substantially expands the set of possible experiments. More importantly, it will greatly simplify the discovery and development of new optogenetic tools.

LED Thermo Flow &amp;#8212; Combining Optogenetics with Flow Cytometry

Optically controlled substances are powerful tools to study signaling pathways. To expand the spectrum of possible experiments, we developed a device for studying optically controlled substances in real time using flow cytometry: the LED Thermo Flow.

LED Thermo Flow - שילוב Optogenetics עם cytometry זרימה

Optogenetic tools allow isolated, functional investigations of almost any signaling molecule within complex signaling pathways. A major obstacle is the ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

הכנה של כרומוזום Meiotic מתפשטת העכבר Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

ניתוח של המתלים תא מבודד מרקמות Solid ידי זרימת cytometry ספקטרלי

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

תא הטרנס-הפנימית המוני הזרקה של תאי גזע עובריים מוביל שיעור גבוה יותר של Chimerism

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can indirectly shape brain function. At the interface between the brain and the systemic circulation, the choroid plexuses (CP), which constitute the blood-cerebrospinal fluid barrier, have been highlighted as a key site of periphery-to-brain communication. CP produce the cerebrospinal fluid, neurotrophic factors, and signaling molecules that can shape brain homeostasis. CP are also an active immunological niche. In contrast to the brain parenchyma, which is populated mainly by microglia under physiological conditions, the heterogeneity of CP immune cells recapitulates the diversity found in other peripheral organs. The CP immune cell diversity and activity change with aging, stress, and disease and modulate the activity of the CP epithelium, thereby indirectly shaping brain function. The goal of this protocol is to isolate murine CP and identify about 90% of the main immune subsets that populate them. This method is a tool to characterize CP immune cells and understand their function in orchestrating periphery-to-brain communication. The proposed protocol may help decipher how CP immune cells indirectly modulate brain function in health and across various disease conditions.

This study uses flow cytometry and two different gating strategies on isolated perfused mice brain choroid plexuses; this protocol identifies the main immune cell subsets that populate this brain structure.

בידוד ואפיון של תאי מערכת החיסון מזכוכית זרחית עכבר מיקרוניתוח

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...