אנו מכירים במימון התמיכה מהמועצה למחקר הבריאות והרפואה הלאומי (להעניק APP605524, 490,037 ו1,047,082), המועצה האוסטרלית למחקר (להעניק DP12010061), האסטרטגיה המחקר המשותפת התשתיות הלאומיות של אוסטרליה ושל קרן החינוך להשקעה מהמחלקה לחדשנות, תעשייה , מדע ומחקר. PML הוא נמען של הפרס שלאחר התואר הראשון באוסטרליה.

כל הנהלים בוצעו בהתאם למדיניות של אוניברסיטת Macquarie והותאמו למועצה הלאומית לבריאות והמחקר רפואי (NHMRC) הקוד אוסטרלי של תרגול. העבודה בוצעה תחת האתיקה הסכם לא ARA 2012/017 אושר והתקבל מוועדת האתיקה בבעלי חיים באוניברסיטת Macquarie. כל הניסויים בוצעו על עכברי SJL / J אלא אם צוין אחרת. 1. עכברים וזיהום ניסיון מלריה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עכברי בית תחת טמפרטורה מבוקרת (21 מעלות צלזיוס) עם מחזור אור-חושך 00:12 שעות. </li><li style=";text-align:right;direction:rtl"> להפשיר aliquot של פ cryopreserved DS Adami chabaudi עם 5% RBCs parasitized (pRBCs) ולהזריק 200 μl לתוך חלל intraperitoneal של C57BL / 6 עכבר תורם. </li><li style=";text-align:right;direction:rtl"> צג parasitemia עכבר תורם כל 1-2 ימים באמצעות cytometry זרימה כפי שתואר בסעיף 3 - 4 לפרוטוקול זה. ברגע שמגיעים לתורמים 5 - parasitemia 15%, לאסוף דם על ידי לנקב לב כדלקמן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl">לשאוב מפתרון הפרין (300 U הפרין / מיליליטר בפתרון העכבר Ringer (MTR) (154 מ&quot;מ NaCl, 5.6 מ&quot;מ KCl, 1 מ&quot;מ MgCl 2, 2.2 מ&quot;מ CaCl 2, 20 מ&quot;מ HEPES 100 μl, 10 גלוקוז מ&quot;מ, 30 U / ml הפרין, pH 7.4, 0.22 מסנן מיקרומטר מעוקר)) לתוך מזרק 1 מיליליטר עם מחט 26 G. </li><li style=";text-align:right;direction:rtl"> להרדים את העכבר על ידי שאיפה עם isofluorane 5%, המאשר את העומק הרצוי של הרדמה על ידי היעדר תגובת נסיגת דוושה ורפלקסים של קרנית. </li><li style=";text-align:right;direction:rtl"> הנח את העכבר על צידו ומחט להכניס ניצב ממש מתחת למרפק, דרך הצלעות, ואל תוך הלב. משוך את בוכנת מזרק באיטיות ובלסובב מחט עד 0.5-1 מיליליטר של דם מתקבל. </li><li style=";text-align:right;direction:rtl"> לבצע המתת חסד הומנית נקע בצוואר רחם בהרדמה. </li></ol></li><li style=";text-align:right;direction:rtl"> לחשב את pRBCs לכל נפח של דם תורם בהנחת ספירה של 9 x 10 6 RBC / μl (דם כלומר, אם התורם היה ב 10% parasitemia כאשר הקריבו, דם יכיל 9 x 10 5pRBC / μl). לדלל דם parasitized בקרבס נאגר מלוח (126 מ&quot;מ NaCl, 2.5 מ&quot;מ KCl, 25 מ&quot;מ NaHCO 3, 1.2 מ&quot;מ לאא 2 PO 4, 1.2 מ&quot;מ MgCl 2, 2.5 מ&quot;מ CaCl 2, גלוקוז 0.2%, pH 7.2, 0.22 מסנן מיקרומטר מעוקר) כך שהריכוז הוא 1 x 10 4 pRBCs ל-200 μl ולהזריק 200 μl לתוך חלל intraperitoneal של עכברים הנדרשים לתיוג RBC וניסוי עירוי. </li><li style=";text-align:right;direction:rtl"> צג parasitemia כל 1-2 ימים באמצעות cytometry זרימה כמתואר בסעיפים 3-4 לפרוטוקול זה. </li></ol> 2. תיוג של RBCs ועירוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> איסוף דם heparinized מעכברים ידי לנקב לב כמתואר בשלב 1.3, ומניח על קרח. לשמור על דם על 4 מעלות צלזיוס בכל העת. לאסוף כ -200 דם μl לכל עכבר להיות מוזרק. במידת צורך, לפצל דם לשתי דגימות ולטפל דגימה אחת כרצוי. הערה: בדרך זו, פלישה של samp טופלניתן להשוות le לשליטה, לדוגמא מטופל (ראה איור 1 עבור סכמטית). </li><li style=";text-align:right;direction:rtl"> הכן פתרון 2x של כל תווית RBC ניאון. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לדלל 2 מ&quot;ג / מיליליטר פתרון מניות של Atto 633-N-Hydroxysuccinimide (Atto 633-NHS) בMTR, כך שהריכוז הוא 20 מיקרוגרם / מיליליטר. </li><li style=";text-align:right;direction:rtl"> לדלל 25 מ&quot;ג / מיליליטר פתרון מניות של ביוטין-N-Hydroxysuccinimide (ביוטין-NHS) בMTR, כך שהריכוז הוא 250 מיקרוגרם / מיליליטר. </li></ol></li><li style=";text-align:right;direction:rtl"> פיצול דגימות דם לשני צינורות ולהוסיף נפח שווה של פתרון 633-NHS 2x Atto לצינור אחד, ונפח שווה של 2x יוטין-NHS לצינור האחר. מערבבים מייד. דגירה דם על 4 מעלות צלזיוס במשך שעה 1 עם ערבוב איטי קבוע, או לחלופין, לערבב מדגם כל 10 - 15 דקות. </li><li style=";text-align:right;direction:rtl"> לשטוף 3 פעמים שכותרתו דם עם MTRC (MTR 0.5% אלבומין + סרום שור (BSA), 0.22 מסנן מיקרומטר מעוקר) כדלקמן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף לפחות 2 כרכים של MTRC, צנטריפוגות ב 750 XG במשך 5 דקות, להסיר את supernatant, וresuspenד גלולה בלפחות 2 כרכים של MTRC. חזור על פי 2 יותר. </li><li style=";text-align:right;direction:rtl"> לאחר לשטוף הסופיים, צנטריפוגות ב 750 XG דקות ו -5 לשלב דם pelleted בשילובים שונים (כלומר, שלא טופל Atto 633 / ביוטין טופל, ביוטין שלא טופל / טופל Atto 633). להוסיף MTR לעשות עד נפח של 200 μl לכל עכבר להיות מוזרקות. </li></ol></li><li style=";text-align:right;direction:rtl"> הזרק דם שכותרתו intravascularly לעכברים נגועים במלריה מכרסם או בקרות נגוע כדלקמן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לבצע עירוי של 2 - parasitemia 15% בשיאו של רְבִיַת שֶׁסַע הטפיל. הערה: זה קורה בערך באמצע הדרך דרך המחזור הכהה (כלומר, בין 12-2 בבוקר במחזור אור רגיל, או 12-2 בערב במחזור אור הפוך). </li><li style=";text-align:right;direction:rtl"> עכברים חמים באמצעות מנורת חום למשך 5-10 דקות כדי להגביר את זרימת דם, אך לנקוט באמצעי זהירות שלא לחמם יותר מדי את העכברים. מניחים עכברים במכשיר הרחקה וintravascularly להזריק 200 μl דם שכותרתו (כ 1 - 2 x 10 RB 9Cs) דרך וריד הזנב. </li></ol></li><li style=";text-align:right;direction:rtl"> לאסוף דגימות דם בנקודות זמן שונות לאחר ההזרקה כפי שתואר בסעיף 3. ניתן לכמת שיעורי פלישה פחות מ -10 דקות לאחר הזרקה, בהתאם לparasitemia של עכבר הנמען: הערה. </li></ol> 3. איסוף דגימות דם והכנה לcytometry הזרימה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 50 μl של פתרון מכתים לדגימה, בתוספת נוספת, ביום של הניסוי כדלקמן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להפשיר aliquot של 6 מ&quot;מ JC-1 המאוחסן ב -20 ° C בsulfoxide דימתיל (DMSO). </li><li style=";text-align:right;direction:rtl"> 50 μl החם של MTRC לדגימה, בתוספת נוספת, עד 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> בעוד ברציפות vortexing MTRC מראש חימם, להוסיף JC-1, כך שהריכוז הסופי הוא 12 מיקרומטר. הדבר נעשה כדי להפחית את היווצרות JC-1 אגרגטים; אם מצרפים אין טופס, לעשות צינור לא צנטריפוגות כמו זה יהיה למנוע הכתמה. הערה: כמות קטנה של צבירה לא תשפיע על תוצאות. </li><li style=";text-align:right;direction:rtl"> להוסיף אנטיCD45 APC eFluor 780 ואנטי-CD71 PerCP eFluor 710, כך שהריכוז הסופי הוא מיקרוגרם / מיליליטר 1. אם ביצוע ניסוי RBC שכותרתו, להוסיף streptavidin PE-Cy7 כך שהריכוז הסופי הוא מיקרוגרם / מיליליטר 1. </li></ol></li><li style=";text-align:right;direction:rtl"> בצע דימום זנב על ידי קטיעה של קצה הזנב או קריעה של כלי הדם בתוך הזנב. מניחים ירידה של דם זנב על שקופית לשקול סירה או זכוכית קטנה. לאחר איסוף דם, לוודא שדימום הפסק. פיפטה 3 μl דם זנב לתוך 50 μl של פתרון מכתים מראש לחמם 37 ° C ו דגירה דגימות ב 37 מעלות צלזיוס למשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> להפשיר aliquot של 4 מ&quot;מ Hoechst 33342 מאוחסן ב -20 ° C במים מזוקקים (אם באמצעות לייזר 355 ננומטר) או 2 מ&quot;מ Hoechst 34580 מאוחסן ב -20 ° C במים מזוקקים (אם באמצעות לייזר 405 ננומטר). הכן 500 μl לדגימה, בתוספת נוספת, של פתרון 4 מיקרומטר או 2 מיקרומטר של Hoechst בMTRC, בהתאמה. </li><li style=";text-align:right;direction:rtl"> הוסף 500 μl של 4 מיקרומטר Hoechst33342 או 2 מיקרומטר Hoechst 34580 לדגימות לדגור על RT במשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> תאי צנטריפוגה ב 750 XG במשך 3 דקות על 4 מעלות צלזיוס, supernatant קלפים שנזרקו, להוסיף 700 μl MTRC ולנתח ידי cytometry זרימה. </li></ol> 4. cytometry הזרימה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ניתוח דגימות באמצעות לייזר 355/488/633 ננומטר או 405/488/633 מכשיר לייזר ננומטר. </li><li style=";text-align:right;direction:rtl"> דגימות מסנן דרך מסננת תא 35 מיקרומטר מייד לפני הניתוח. יש לשטוף את מסננת תא במים מזוקקים בין דגימות ושימוש חוזר. </li><li style=";text-align:right;direction:rtl"> להקליט מספיק אירועים, כך שהאוכלוסייה הקטנה ביותר מכילה לפחות 500 אירועים (בדרך כלל 1,000,000 - 10,000,000 אירועים הכולל לדוגמה). </li><li style=";text-align:right;direction:rtl"> Excite Hoechst 33342 באמצעות לייזר 355 nm ולזהות אירועים דרך מסנן 460/50. Excite Hoechst 34580 באמצעות לייזר 405 nm ולזהות אירועים דרך מסנן 460/50. Excite JC-1, אנטי-CD71 PerCP eFluor 710 וstreptavidin PE-Cy7 באמצעות לייזר 488 ננומטר ולזהות אירועים דרך 530/40, 692/40, ו750LP לסנן בהתאמהly. Excite Atto 633 ואנטי-CD45 APC eFluor 780 באמצעות לייזר 633 ננומטר ולזהות אירועים דרך מסנן 670/30 או 750LP בהתאמה. </li><li style=";text-align:right;direction:rtl"> ביצוע ניתוח ופיצוי באמצעות זרימה מתאימה cytometry תוכנת ניתוח כדלקמן: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בחר תאים שלמים ולא לכלול רעש, פסולת, וטסיות דם המבוססת על FSC מאפיינים / SSC (איור 2 א). בחר תאים בודדים המבוססים על או רוחב דופק הדק (איור 2) או על ידי שימוש באזור שיא FSC יחס גובה (איור 2 ג). </li><li style=";text-align:right;direction:rtl"> בחר RBCs הבוגר, ולא לכלול אבות RBC וכדוריות דם לבנות, המבוסס על הקרינה שלילית בPerCP eFluor 710 וAPC eFluor 780 הערוצים (איור 2 ד). </li><li style=";text-align:right;direction:rtl"> אם ביצוע ניסוי RBC המסומן, בחר כל אוכלוסייה של RBCs שכותרתו מבוסס על PE-Cy7 וAtto 633 הקרינה ולנתח בנפרד (איור 3 א) אלה. </li><li style=";text-align:right;direction:rtl"> בחר pRBCs המבוסס על הקרינה חיובית עבור שני JC-1 וHoechst ( <strאונג&gt; איור 2E-H). </li></ol></li></ol> 5. חישובים וסטטיסטיקות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחשב parasitemia על ידי חלוקת מספר pRBCs במספר הכולל של תאי דם אדומים (כלומר, מספר האירועים בQ2 שער מחולק במספר האירועים בG4 שער). בעת ביצוע parasitemia assay הפלישה של שתי האוכלוסיות שכותרתו יכולה להיות מחושב על ידי חלוקת מספר pRBCs שכותרתו במספר RBCs שכותרתו באוכלוסייה ש( כלומר, מספר האירועים שנמצאים בשני L1 השערים וQ2, מחולק במספר האירועים בשער L1). הערה: parasitemia תווית משתנה במידה ניכרת בהתאם לגורמים כגון parasitemia ומספר merozoites במחזור אנדוגני. מסיבה זו, כדאי לדווח על יחס parasitemia, המחושב כparasitemia של המטופלים האוכלוסייה שהכותרת מחולקת בparasitemia של אוכלוסיית השליטה שכותרתו בכל עכבר בודד. כדי לתקן את השפעות צבע אפשריות, דו&quot;ח תוצאותזה יחס parasitemia ממוצע על פני שני שילובי הצבע. </li><li style=";text-align:right;direction:rtl"> לקבוע מובהקות סטטיסטיות של יחסים באמצעות דגימה אחת -test t עם 1 כממוצע היפותטי. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RBC+barcoding+allows+for+the+study+of+erythrocyte+population+dynamics+and+P.+falciparum+merozoite+invasion.">RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion.</a> PLoS One. 9 (7), e101041 (2014).</li><li>Malleret, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+robust+tri-color+flow+cytometry+assay+for+monitoring+malaria+parasite+development.">A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.</a> Sci Rep. 1 (118), (2011).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+measurement+of+Plasmodium-infected+erythrocytes+in+murine+models+of+malaria+by+flow+cytometry+using+bidimensional+assessment+of+SYTO-16+fluorescence.">Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence.</a> Cytometry A. 75 (3), 225-235 (2009).</li><li>Jimenez-Diaz, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+of+detection+specificity+of+Plasmodium-infected+murine+erythrocytes+by+flow+cytometry+using+autofluorescence+and+YOYO-1.">Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1.</a> Cytometry A. 67 (1), 27-36 (2005).</li><li>Jun, G., Lee, J. S., Jung, Y. J., Park, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+Plasmodium+parasitemia+by+flow+cytometry+and+microscopy.">Quantitative determination of Plasmodium parasitemia by flow cytometry and microscopy.</a> J Korean Med Sci. 27 (10), 1137-1142 (2012).</li><li>Lelliott, P. M., Lampkin, S., McMorran, B. J., Foote, S. J., Burgio, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometric+assay+to+quantify+invasion+of+red+blood+cells+by+rodent+Plasmodium+parasites+in+vivo.">A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.</a> Malar J. 13 (1), 100 (2014).</li><li>Morohashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+spleen+of+an+mFtz-F1+gene-disrupted+mouse.">Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.</a> Blood. 93 (5), 1586-1594 (1999).</li><li>Shet, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+functional+platelet+abnormalities+in+Berkeley+sickle+cell+mice.">Morphological and functional platelet abnormalities in Berkeley sickle cell mice.</a> Blood Cells Mol Dis. 41 (1), 109-118 (2008).</li><li>Duraisingh, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variation+of+Plasmodium+falciparum+merozoite+proteins+directs+receptor+targeting+for+invasion+of+human+erythrocytes.">Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes.</a> EMBO J. 22 (5), 1047-1057 (2003).</li><li>Okoyeh, J. N., Pillai, C. R., Chitnis, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+field+isolates+commonly+use+erythrocyte+invasion+pathways+that+are+independent+of+sialic+acid+residues+of+glycophorin+A.">Plasmodium falciparum field isolates commonly use erythrocyte invasion pathways that are independent of sialic acid residues of glycophorin A.</a> Infect Immun. 67 (11), 5784-5791 (1999).</li><li>Dolan, S. A., Miller, L. H., Wellems, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+switching+mechanism+in+the+invasion+of+erythrocytes+by+Plasmodium+falciparum.">Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.</a> J Clin Invest. 86 (2), 618-624 (1990).</li></ol>

יש לי המחברים אין לחשוף.

יש לנו תיארתי שיטה למדידה של שתי פלישת parasitemia וmerozoite של דגימות in vivo. במונחים של מדידת parasitemia, שיטה זו מציעה יתרון על פני שיטות קודמות 10-13 שבHJ-RBCs ניתן להבחין בין pRBCs, ובכך להפחית את מספר האירועים חיוביים כוזבים. בעוד HJ-RBCs הוא בדרך כלל נדיר בבני אדם, כמה מחקרים מדו...

The clinical symptoms associated with malaria occur during the Plasmodium parasite&#8217;s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs. 
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia. 
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td>Hoechst 33342. Store a 4mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>Hoechst 34580</td>
			<td>Sigma-Aldrich</td>
			<td>63493</td>
			<td>Store a 2mM stock solution at -20 &#176;C in distilled water</td>
		</tr>
		<tr>
			<td>JC-1 Dye</td>
			<td>Life Technologies</td>
			<td>T-3168</td>
			<td>Store small aliquots of 6mM stock solution at -20 &#176;C in DMSO</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD45 APC-eFluor 780</td>
			<td>eBioscience</td>
			<td>47-0451-80</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD71 PerCP-eFluor 710</td>
			<td>eBioscience</td>
			<td>46-0711-80</td>
			<td>Clone R17217</td>
		</tr>
		<tr>
			<td>Atto 633 NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>1464</td>
			<td>Atto 633-NHS. Store a 2mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>EZ-Link Sulfo-NHS-LC-Biotin</td>
			<td>Thermo Fisher Scientific</td>
			<td>21335</td>
			<td>Biotin-NHS. Store a 25mg/ml stock solution at -20 &#176;C in DMF</td>
		</tr>
		<tr>
			<td>Streptavidin PE-Cyanine7</td>
			<td>eBioscience</td>
			<td>25-4317-82</td>
			<td>Streptavidin PE-Cy7</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>H478</td>
			<td></td>
		</tr>
		<tr>
			<td>35&#181;M filter cap tubes</td>
			<td>Becton Dickinson</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD LSRFortessa</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD FACSAria II</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: BD Influx</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer: CyAn ADP Analyzer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo assessment of rodent plasmodium parasitemia and merozoite invasion by flow cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

מדידה של parasitemia. למדידה parasitemia, צריכים להיות ראשון שנבחרו תאי דם, ורעש, פסולת וטסיות דם נשללו, המבוססים על מאפייני FSC / SSC (איור 2 א). בהתאם לcytometer משמש, אז צריכים להיות נבחרו תאים בודדים המבוססים על או רוחב דופק הדק (איור 2),</s...

Watch this Scientific Journal Video about In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry at JoVE.com

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

פולש טפיל המלריה והמשכפל בתוך תאי דם אדומים. ההערכה מדויקת של פלישת merozoite וparasitemia לכן חיונית בהערכה במהלך הידבקות במלריה. כאן אנו מתארים זרימה cytometry פרוטוקול מבוסס למדידת הפרמטרים הללו במודל עכבר של מלריה.

בVivo ההערכה של מכרסמים Plasmodium Parasitemia וMerozoite פלישה על ידי cytometry הזרימה

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

in-vivo-assessment-rodent-plasmodium-parasitemia-merozoite-invasion

Research

JoVE Journal

Immunology and Infection

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

9.5K Views.  Macquarie University.  פולש טפיל המלריה והמשכפל בתוך תאי דם אדומים. ההערכה מדויקת של פלישת merozoite וparasitemia לכן חיונית בהערכה במהלך הידבקות במלריה. כאן אנו מתארים זרימה cytometry פרוטוקול מבוסס למדידת הפרמטרים הללו במודל עכבר של מלריה. 

Video: In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

מדידת פעילות Calpain בתוך תאים חיים קבוע על ידי זרימת cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

הערכה כמותית של תאים חיסוניים רקמות חוט השדרה נפגע על ידי זרימת cytometry: השתמש חדשנית שיטת טיהור Cell

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

אנושי במבחנה דיכוי ככלי מיון עבור הכרה של מדינת מוקדם של חוסר איזון החיסון

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

בחינה של בחירה חיובית ושלילית הרתי ידי זרימת cytometry

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

This manuscript describes a detailed protocol for phenotypic and quantitative analysis of resident macrophages from rat kidneys by flow cytometry. The resulting stained cells can be also used for other applications, including cell sorting, gene expression analysis or functional studies, thus increasing the information obtained in the experimental model.

אפיון פנוטיפי של מקרופאגים כליה עכברוש ידי cytometry זרימה

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

להערכת תפקוד רשתית microglial Phagocytic<em&gt; In vivo</em&gt; שימוש cytometry זרימה מבוסס Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

ניתוח תזרים Cytometric נ בית נכנס חלקיקים 1 האלרגנים PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

בידוד והתרבות במבחנה של מקרופאגים מכתשי מאתר ואנושי

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

אפיון של קבוצות משנה מונוציט האנושית על ידי דם לזרום Cytometry ניתוח

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. Setup of a classic multicolor panel requires high-end instruments, custom labeled antibodies, and careful study design to minimize spectral overlap. We developed a multiparametric analysis to identify major human immune populations (CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes) in peripheral blood by combining seven lineage markers using only two fluorochromes. Our strategy is based on the observation that lineage markers are constantly expressed in a unique combination by each cell population. Combining this information with a careful titration of the antibodies allows investigators to record five additional markers, expanding the optical limit of most flow cytometers. Head-to-head comparison demonstrated that the vast majority of immune cell populations in peripheral blood can be characterized with comparable accuracy between our method and the classic &#34;one fluorochrome-one marker approach&#34;, although the latter is still more precise for identifying populations such as NKT cells and &#947;&#948; T cells. Combining seven markers using two fluorochromes allows for the analysis of complex immune cell populations and clinical samples on affordable 6-10 fluorochrome flow cytometers, and even on 2-3 fluorochrome field instruments in areas with limited resources. High-end instruments can also benefit from this approach by using extra fluorochromes to accomplish deeper flow cytometry analysis. This approach is also very well suited for screening several cell populations in the case of clinical samples with limited number of cells.

Here, we present a flow cytometric protocol to identify CD4+ and CD8+ T cells, &#947;&#948; T cells, B cells, NK cells and monocytes in human peripheral blood by using only two fluorochromes instead of seven. With this approach, five additional markers can be recorded on most flow cytometers.

אפליה של שבע קבוצות משנה תא החיסון על ידי שני-fluorochrome לזרום Cytometry

Immune cell characterization heavily relies on multicolor flow cytometry to identify subpopulations based on differential expression of surface markers. ...