אנו מודים לד&quot;ר Vladana Vukojeviç, מרכז לרפואה מולקולרית, קרולינסקה לשמירה על המכשיר Zeiss Confocor 3 ועבור עצות מועילות לגבי מדידות תא. מחקר זה מומן על ידי מענקים Vetenskapsrådet (מספר מענק 2012- 1629), מגנוס Bergvalls stiftelse, ומן Stiftelsen קלאס Groschinskys minnesfond.

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The authors have nothing to disclose.

פרוטוקול זה עבור FCS יכול לשמש להערכת דינאמיקה מולקולארית של מולקולות פני השטח על כל סוגי תאים חיסוניים (Murine, אדם, או מינים אחרים). אמצעי FCS במרחב ובזמן דינאמיקה מולקולארית עד רזולוצית מולקולה בודדת בתאים חיים. הצפיפות המולקולרית, וכן את דינמיקת קצב אשכולות דיפוזיה של ה...

פונקציות תאים חיסוניים רבות מסתמכות על דיפוזיה אינטראקציות מולקולריות בתוך ממברנות. ממברנות ביולוגיות הם מורכבים, וישנם גורמים רבים שעשויים להיות חשובים לתפקוד של תאי מערכת החיסון יכול להשפיע על מהירות של דיפוזיה translational של חלבונים בתוך קרומים תאיים 1. הראינו לאחרונה כי הרג טבעי (NK) תאים, לימפוציטים שייך למערכת החיסון המולדת, להפגין דיפוזיה הפרש של שני חלבונים למדו בבית קרום התא בהתאם למצב של תא NK הפעלה 2. מתאם ספקטרוסקופיה פלואורסצנטי (FCS) היא טכניקה כי הוא מסוגל לכימות שיעורי דיפוזיה מולקולרית בתוך ממברנות ביולוגיות. זה מדווח על קצב הדיפוזיה הממוצע של fluorescently שכותרתו מולקולות בנפח קבוע, בדרך כלל מוקד מיקרוסקופ confocal. היא מבוססת על מדידת תנודות קרינה המתרחשת על תנועה מולקולרית במערכת במצב עמיד. FCS כבר בשימוש נרחב ללימוד דיפוזיה של צבעי ניאון וחלבונים, הוא פתרון ובתוך ממברנות שומנים בדם. פרמטרי פלט נוספים המשפיעים על קצב הדיפוזיה יכולים להיות גם למדו בעקיפין בדרך זו (למשל, שינויים קונפורמציה של חלבונים או אינטראקציות של מולקולות על קרום תא) 3, 4. FCS בולטת לעומת טכניקות אחרות בשל הרגישות הגבוהה שלו, מה שמאפשר את האפשרות לגילוי מולקולה בודדת. זה עובד גם עבור ריכוזים מולקולריים nanomolar למגוון millimolar, אשר אופיינית עבור רמות ביטוי אנדוגני של רוב החלבונים 5. יתר על כן, FCS יכול לתת קירוב המספר המוחלט של חלבונים בתוך הנפח למד, בעוד רוב הטכניקות אחרות רק לתת מידע ביחס על רמות ביטוי חלבון. שיטות אחרות כדי למדוד את שיעורי דיפוזיה מולקולרית בתוך ממברנות כוללים fluoהתאוששות rescence לאחר photobleaching (FRAP), חלקיק בודד מעקב (SPT), FCS חריר המרובה, ושיטות התמונה קורלציה. FRAP ושיטות תמונת מתאם טכניקות הרכב, אשר בדרך כלל לא מספקות מידע על מספרם המוחלט של מולקולות 10. לעומת SPT, התפוקה של FCS היא גבוהה ביחס המאפיין את הממוצע באוכלוסייה. הניתוח הוא גם פחות תובעני מאז קצב הדיפוזיה הממוצע של המולקולות הנוכחות בתוך מוקד הליזר נמדד, ולא בשיעור של מולקולות בודדות. כמו כן, אלא אם כן מיקרוסקופים מיוחדים זמינים 11, SPT לא יכול לתת שום מידע על ריכוזי, מאז תיוג SPT תקן חייב להיות נמוך מאוד על מנת לאפשר זיהוי של מולקולות בודדות. מצד השני, FCS מחייב המולקולות הנחקרת להיות ניידות. זה פשוט לא מזהה כל שברים המשוערת נייחים או מולקולות נעו לאט יותר. שיעור דיפוזיה של מולקולותמתגוררים בתוך המוקד שאורכן עולה על עשירית מהזמן הרכישה לא להיות מיוצג כראוי במדידות FCS 3, 12. לכן, מקדמי דיפוזיה שרשמו FCS נוטים להיות מהיר יותר מאשר שיעורי דיפוזיה דיווחו מטכניקות כמו FRAP ו SPT, שבו קרוב ל-נייח איטית מאוד שברים נלקחים בחשבון גם כן. SPT גם ייתן תיאור מפורט יותר של ההשתנות של שיעורי דיפוזיה בתוך האוכלוסייה המולקולרית מ FCS יהיה. FCS מכמת את התנודות של עוצמת קרינה לאורך זמן בתוך הנפח הנרגש. במקרה של מדידות קרום, זה מתרגם ל השטח המואר של הממברנה. במאמר זה, אנו מנצלים את העובדה כי תנודות כאלה הן המושרה על ידי מולקולות המפגינות דיפוזיה בראונית ובכך עוברות פנימה וחוצה של נפח העירור. ישנם גם מספר מקורות אפשריים אחרים עבור fluctuaהמשא ב האות הקרינה, כגון מהבהב או נוכחות של טריפלט ב fluorophores, השפעות סביבתיות, מחייב unbinding של ליגנד, או תנועה של קרום התא כולו. מקורות שגיאה המשוערים אלה צריכים להילקח בחשבון בעת תכנון ניסוי FCS כדי לפרש את התוצאות במדויק 12, 13. בדרך כלל, שיעורי דיפוזיה לרוחב ממברנות ביולוגיות נמוכים בשל צפיפות ואינטראקציות, הוא בין חלבונים בממברנה ובין חלבוני שלד התא. מבחינה הסטורית, השימוש FCS בממברנות ובכך כבר הקשה על ידי חוסר fluorophores צילום יציב, אשר נדרשים כדי למנוע הלבנה בזמני המעבר המורחבים דרך התמקדות עירור 14. עם זאת, היום, יש שפע של אפשרויות עבור צבעי צילום יציב מתאימים. שיפורים משמעותיים גלאי ורכיבי חומרה אחרת גם לאפשר זיהוי של prote הניאוןכל פרטי צבעים של בהירות נמוכה יותר. כאן, פרוטוקול בסיסי עבור היישום של FCS באמצעות לימפוציטים העיקרי בעכברים, שבו החלבון של עניין מסומן עם fluorescently מתויג נוגדן, מתואר. גישה כדי להתאים את עקומות autocorrelation כדי לחלץ את מקדם הדיפוזיה ואת הצפיפות המולקולרית מוצגת גם. הפרוטוקול שיתפשר עוקב אחריו בקלות על ידי אימונולוגים ללא ניסיון קודם של טכניקות ללמוד דיפוזיה של מולקולות. עם זאת, הבנה בסיסית של מיקרוסקופיה confocal צפויה (כדי לקבל הבנה בסיסית זו, ראה התייחסות 15). פרוטוקול זה יכול להיות מותאם בקלות יחסית לתאי השעיה אחרים, הוא שורות תאי תאים ראשוניים. עבור משתמשים מנוסים יותר FCS, שיטות ניתוח מעודנות יותר להתקיים, שחלקם מתוארים הדיון.

<table border="0" cellpadding="0" cellspacing="0" width="1206">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">MACS NK Cell Isolation Kit mouse II</td>
			<td style="width:367px;">Miltenyi Biotec NordenAB</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:337px;">Negative selection of NK cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Fetal Bovine Serum</td>
			<td style="width:367px;">Sigma-Adlrich</td>
			<td style="width:181px;">F7524</td>
			<td style="width:337px;">Heat inactivated</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Phosphate Buffered Saline</td>
			<td style="width:367px;">-</td>
			<td style="width:181px;">-</td>
			<td style="width:337px;">Made in house&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Roswell Park Memorial Institute medium 1640</td>
			<td style="width:367px;">PAA The Cell Culture Company&#160;</td>
			<td style="width:181px;">E15-848</td>
			<td style="width:337px;">Transparent medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Antibody clone 2.4G2</td>
			<td style="width:367px;">Thermo Fischer Scientific</td>
			<td style="width:181px;">553140</td>
			<td style="width:337px;">For blocking Fc-receptors.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-Ly49A antibody&#160;</td>
			<td rowspan="2" style="width:367px;"></td>
			<td rowspan="2" style="width:181px;"></td>
			<td rowspan="2" style="width:337px;">Monoclonal antibody made in house and conjugated in house to Alexa fluor 647</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Clone JR9.318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-H-2Dd antibody&#160;</td>
			<td rowspan="2" style="width:367px;">BD Pharmingen</td>
			<td rowspan="2" style="width:181px;">558915</td>
			<td rowspan="2" style="width:337px;">Conjugated in house to MFP488</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Clone 34.5.8S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">MFP488</td>
			<td style="width:367px;">Mobiotech</td>
			<td style="width:181px;">MFP-A2181</td>
			<td style="width:337px;">Fluorescent dye for antibody conjugation.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Poly-L-Lysine</td>
			<td style="width:367px;">Sigma-Aldrich</td>
			<td style="width:181px;">P8920</td>
			<td style="width:337px;">Diluted in distilled water (1.10)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:321px;">Poly-L-Lysine (20 kDa) grafted with polyethylene glycol (2 kDa)</td>
			<td style="width:367px;">SuSoS AG</td>
			<td style="width:181px;">PLL(20)-g[3.5]-PEG(2)</td>
			<td style="width:337px;">Diluted in PBS (pH 7.4) to 0.5 mg/ml.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">Rhodamine 110 chloride</td>
			<td style="width:367px;">Sigma-Aldrich</td>
			<td style="width:181px;">432202</td>
			<td style="width:337px;">Known diffusion coefficient: 3.3 &#215; 10&#8722;10 m2/s 19</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:321px;">Alexa fluor 647</td>
			<td style="width:367px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:181px;">A20006</td>
			<td style="width:337px;">Known diffusion coefficient: 4.4 &#215; 10&#8722;10 m2/s 20</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Confocal microscope&#160;</td>
			<td style="width:367px;">Zeiss</td>
			<td style="width:181px;">LSM510</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Software: Confocor 3</td>
			<td style="width:367px;">Zeiss</td>
			<td style="width:181px;"></td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Software: Matlab with curve fitting toolbox</td>
			<td style="width:367px;">Matlab</td>
			<td style="width:181px;">Version R2013b</td>
			<td style="width:337px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:321px;">Nunc Lab-Tek Chambered Coverglass</td>
			<td style="width:367px;">Thermo-scientific&#160;</td>
			<td style="width:181px;">155411</td>
			<td style="width:337px;">8 wells, 1.0 borosilicate bottom</td>
		</tr>
	</tbody>
</table>

molecular diffusion in plasma membranes of primary lymphocytes measured by fluorescence correlation spectroscopy

מתאם ספקטרוסקופיה פלואורסצנטי (FCS) היא טכניקה רבת עוצמה ללימוד דיפוזיה של מולקולות בתוך ממברנות ביולוגיות עם רזולוציה גבוהה במרחב ובזמן. FCS יכול לכמת את מקדם ריכוז דיפוזיה מולקולרית של מולקולות שכותרתו fluorescently בקרום התא. טכניקה זו יש את היכולת לחקור את מאפייני דיפוזיה המולקולריים של מולקולות בקרום הפלזמה של תאים חיסוניים מצב יציב (כלומר, בלי תהליכים המשפיעים על התוצאה בזמן המדידה בפועל). FCS מתאים ללימוד דיפוזיה של חלבונים כי הם לידי ביטוי ברמות אופייניות חלבונים אנדוגניים ביותר. כאן, שיטה פשוטה וחזקה כדי לקבוע את קצב הדיפוזיה של חלבוני קרום על לימפוציטים עיקרי באה לידי ביטוי. דרך יעילה לבצע מדידות על תאי חיים מוכתם נוגדן נפוץ תצפיות שהתרחשו לאחר הרכישה מתוארת. הפיתוחים האחרוניםבפיתוח של צבעי ניאון צילום יציב יכול להיות מנוצל על ידי conjugating הנוגדנים של עניין לנכס צבעים שאינם להלבין בהרחבה במהלך המדידות. בנוסף, זה מאפשר זיהוי של גופים להתפוגג אט אט, אשר היא תכונה נפוצה של חלבונים הביע קרום התא. הליך הניתוח כדי לחלץ פרמטרי ריכוז דיפוזיה מולקולריים מן עקומות autocorrelation שנוצרו מודגש. לסיכום, פרוטוקול בסיסי למדידות FCS מסופק; ניתן בעקבותיו ידי אימונולוגים עם הבנה של מיקרוסקופיה confocal אך ללא ניסיון קודם אחרים של טכניקות למדידת פרמטרים דינמיים, כגון שיעורי דיפוזיה מולקולרית.

תוצאה אופיינית יפיק עקומת autocorrelation עם זמן המעבר בטווח של 10 מילי-שניות ל -400 מילי-שניות עבור חלבונים בממברנה. מספר מולקולות יכול להשתנות בין 0.5 ל סביב 200 לכל מיקרומטר 2 עבור חלבונים הביע באופן אנדוגני. בדוק היטב כי CPM אינו נמוך מהצפוי. המשמעות יכולה...

Watch this Scientific Journal Video about Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy at JoVE.com

דיפוזיה מולקולרית בממברנות פלזמה של לימפוציטים ראשוניים נמדדת באמצעות ספקטרוסקופיה של מתאם פלואורסצנטי | אימונולוגיה וזיהום | וִידֵאוֹ

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

דיפוזיה מולקולרית פלזמה ממברנות יסודי לימפוציטים נמדד באמצעות מתאם ספקטרוסקופיה פלואורסצנטי

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

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10.8K Views.  Karolinska Institutet. A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Video: Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

שיטות להעריך מתווכת בטא מוות של תאים על ידי לימפוציטים מסוג T ציטוטוקסיות

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

שינוי מערכת Transposon piggyBac של תאי T אנושיים ראשוניים

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

ניתוח מהיר של סטיות כרומוזום בעכבר לימפוציטים מסוג B על ידי הרשות הפלסטינית-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

כימות של Cytosolic לעומת vacuolar<em&gt; סלמונלה</em&gt; ביסודי המקרופאגים על ידי דיפרנציאל permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

מדידת pH Phagosome ידי ratiometric מיקרוסקופ פלואורסצנטי

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

קליפת המוח אקטין זרימה בתאי T לכמת ידי מתאם תמונת ספקטרוסקופיה מרחב ובזמן של תאורה מיקרוסקופית נתונים מובנים

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

הגירה ו מהירות Phagosome נמדד המקרופאגים אדם מן המעלה הראשונה חי עם HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

ויזואליזציה של לימפוציטים-22-להביע IL באמצעות עכברים Reporter

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

לימפוציטים בידוד מן העכבר קטן המעי המערכת החיסונית

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

כימות של Efferocytosis על-ידי קרינה פלואורסצנטית תא בודד מיקרוסקופ

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...