المؤلفان بالشكر خوسيه لويس دي لا فيغا، اريكا Melchy والدكتور تاكويا نيشيجاكي للحصول على المساعدة الفنية. وأيد هذا العمل من قبل المجلس الوطني للبحوث والتكنولوجيا Ciencia (CONACYT والمكسيك) (99333 و 128566 لCT)؛ المديرية العامة للAsuntos ديل الشخصية Académico / جامعة المكسيك الوطنية المستقلة (IN202212-3 إلى CT).

<ol>
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ليس لدينا شيء في الكشف عنها.

إشارات بين الخلايا هو أمر حيوي لمعظم الأنشطة الخلوية؛ كا 2 + هو رسول في كل مكان التي ترافق خلايا الثدييات في جميع أنحاء كامل حياتها، من أصلهم في التسميد، إلى نهاية دورة حياتها. في استجابة للمؤثرات مختلفة، [كا 2 +] ط الزيادات، يتذبذب والنقصان مع تدوين المكاني?...

كا 2 + هو رسول الثاني العالمي لمسارات نقل الإشارة في الخلايا حقيقية النواة. داخل الخلايا كا 2 + (كا 2 + ط) يشارك في تنظيم العديد من العمليات الفسيولوجية الأساسية في الخلايا على حد سواء منفعل وغير منفعل. مشتق أهمية وعالمية كا 2 + كرسول الثانية خلال الأحداث نقل الإشارة من تنوعها المكانية والزمانية في نقل المعلومات داخل الخلية. بينما كا 2 + لا يمكن توليفها من جديد أو المتدهورة داخل الخلية، وتركيزه داخل الخلايا ([كا 2 +] ط) يتم الاحتفاظ ضمن حدود صارمة للغاية من خلال الآليات الخلوية المختلفة التي العازلة بشكل مستمر، تنحية، تجزئة، و / أو تتراكم كا 2 +. يمكن أن تحدث تغييرات في تركيز أيون في هذه المناطق المترجمة للغاية داخل الخلية 1، وفك رموز هذه التقلبات أمر ضروري لاكتساب ديفهم EPER من (1) دورهم في آلية يشير، (2) أهميتها الفسيولوجية، و (3) آليات العام للإشارة الخلية. كا 2 + إشارات بوساطة له أهمية خاصة في علم وظائف الأعضاء الحيوانات المنوية 2. الحيوانات المنوية على الحركة هي واحدة من أهم وظائف للنجاح الإخصاب، في واقع الأمر، يمكن أن العديد من العيوب الحيوانات المنوية على الحركة يسبب العقم 3-5. أهمية الكالسيوم 2 + في حركة سوطي وقد أدركت منذ فترة طويلة 6، ولكن آلية لكيفية كا 2 + يتحكم في شكل محدد من أشكال سوطي الانحناء ليست مفهومة تماما. قبل التفجير مع البويضة، يجب أن تخضع الحيوانات المنوية وتمكين الطاقات، وهي عملية معقدة تعتمد على الإقامة الحيوانات المنوية داخل المسالك الإناث. خلال تمكين الطاقات، يتم تعديل بنية الدهون غشاء الحيوانات المنوية ومنظمة، وذلك أساسا نتيجة لإزالة الكوليسترول من غشاء البلازما. بالإضافة إلى ذلك، العديد من البروتينات هي التيروزين-الفوسفورylated 7. الأهم من ذلك، خلال تمكين الطاقات هناك زيادة في درجة الحموضة داخل الخلايا (درجة الحموضة ط) و في [كا 2 +] ط، وhyperpolarizes غشاء المحتملة في بعض الأنواع 2. تمكين الطاقات يستغرق سوى مكان في جزء من السكان من الحيوانات المنوية (20-40٪)، والآليات التي تشارك في كل هذه التغيرات الخلوية لا تزال بعيدة عن الوضوح. ومن المسلم به عموما أن فقط جزء من السكان من الحيوانات المنوية باكتسابها الخضوع لتفاعل الجسيم الطرفي (AR) عندما تتعرض لفائف الفسيولوجية. ع هو أيضا كا 2 + حدث التنظيم المطلوب للإخصاب في جميع الأنواع امتلاك الجسيم الطرفي (عضية المتخصصة مع الأغشية الخارجية والداخلية). خلال هذه العملية الصمامات غشاء الجسم الطرفي الخارجي مع غشاء البلازما المنوية، والإفراج عن الانزيمات حلمهي التي تسمح للخلية الحيوان المنوي على اختراق مصفوفة غليكو-البروتينية المحيطة البيض (المنطقة الشفافة، أو ZP). أيضا يعرض AR جديدة fusogenic الحيوانات المنوية سطح الخلية التي تتفاعل معغشاء البلازما البيض للانصهار النهائي كل من الأمشاج. هناك عدة بروابط الخلوية التي تحفز AR، البروجسترون كونها واحدة من أكثر درس فيما بينها. في هذا العمل نقدم أربع تقنيات مختلفة تنطوي على استخدام كا 2 صبغة الفلورسنت حساسة + لقياس [كا 2 +] ط التغيرات في الحيوانات المنوية البشرية الناجمة عن البروجسترون (باستثناء التدفق الخلوي، الذي قمنا بقياس [كا 2 + ] أنا يسببها زيادة خلال عملية تمكين الطاقات في المختبر). في هذه الحالة بالذات كنا فلوو-3 AM (الحياة تكنولوجيز، وغراند ايلاند، نيويورك)، صبغة نفاذية الغشاء مع K د = 325 نانومتر. في المختبر فإننا رصد التغيرات مضان بوصفها وظيفة من الوقت مع ثلاثة من المنهجيات، ومع تقنية الرابع قمنا بقياس القيم مضان في نقطة واحدة معينة من الزمن. هذه المقاربات المختلفة تكمل بعضها البعض، تماما منذ أنها توفر الدقة المكانية والزمانيةolution في كل من خلية واحدة ومستويات السكان الخلية. السكان الخلية أو التجارب السائبة وتستخدم تقنيات السائبة على نطاق واسع ليس فقط لأن الصكوك التي تتطلب متوفرة بسهولة، ولكن أيضا لأنها بسيطة، راسخة، والسماح للفي المتوسط ​​من المعلومات من القياسات التي أجريت على ملايين الخلايا في تجربة واحدة. تقنية # 1. قياس التألق التقليدية هذه التقنية تراقب التغيرات في مضان بوصفها وظيفة من الزمن؛ يتم تنفيذ التجارب في cuvettes الزجاج مع حجم العينة تتراوح ما بين 200 إلى 1،000 ميكرولتر. الخلط السليم من الكواشف وأضاف يتطلب التحريك المغناطيسي، وبالتالي فإن القرار الزماني حصلنا عليها في ترتيب ثواني. نموذجية خلية تركيز مجموعة من العينات التي تم تحليلها هي 10 5 -10 8 خلية / مل. تقنية # 2. توقف تدفق قياس التألق Tأسلوبه أيضا تراقب التغيرات في مضان بوصفها وظيفة من الوقت، ولكن الكواشف بسرعة تختلط معا (باستخدام ضغط) في كفيت تسجيل يحتوي على حجم عينة صغيرة جدا (تتراوح 25-100 ميكرولتر). ولذلك، تجانس الكواشف بشكل فوري، مما يتيح قرار زمنية عالية في ترتيب ميلي ثانية. تحليل مضان مقابل الوقت مما آثار هي مناسبة لتحديد معدلات التفاعل، توضيح مدى تعقيد آلية رد الفعل، والحصول على معلومات عن رد فعل وسيطة لم يدم طويلا، وما إلى ذلك مجموعة تركيز خلية مشتركة من العينات التي تم تحليلها هي 10 5 -10 7 خلية / مل. تجارب خلية واحدة تجارب السائبة تقرير السلوك متوسط ​​لعدد كبير من الخلايا، ومع ذلك، قد يبلغ عدد سكانها تظهر في كثير من الأحيان الخصائص غير المتجانسة التي يتم التغاضي عنها خلال هذا النوع من القياسات. وبالتالي تستخدم تقنيات خلية واحدة لتكمل المعلومات البريد التي تم الحصول عليها مع التجارب السكان الخلية. تقنية # 3. التدفق الخلوي وعلى الرغم من أهمية المعلومات الناتجة عن القياسات خلية واحدة، فمن المهم لتحليل عدد كبير من الخلايا من أجل منع استقراء الخاطئة من خصائص خلية محددة لشعب بأكمله. لهذا السبب، ويفضل التقنيات الإنتاجية العالية والأسلوب الأكثر شعبية هو التدفق الخلوي، التي يتم تحليلها في تقليديا 10،000 خلية لكل حالة. تمكن هذه الطريقة تحليل متعدد حدودي من السكان غير متجانسة لأنها تصنف الخلايا وفقا لحجمها (مبعثر إلى الأمام (FSC))، تحبب (مبعثر الجانب (SSC)) وكثافة مضان (وضع العلامات المحددة مع الأجسام المضادة، علامة السلامة، الخ.) ، وبالتالي توفير المعلومات عن توزيع المعلمات &quot;لمجموعة من الخلايا. التدفق الخلوي يوفر الفوري بدلا من المعلومات التي تعتمد على الوقت 8. إلى الأمام والجانب مبعثر القيم عيجب أيضا أن يدرج ه مفيدة أيضا لاختيار البوابة التي تشمل الخلايا ولكن يميز الحطام الخلوية، والغبار، وغيرها للحصول على قياسات مضان، مضان ضوابط السلبية والإيجابية. إذا تم استخدام قناة مضان أكثر من واحد، وهي عملية تعرف باسم التعويض يجب أن يؤديها (لمزيد من التفاصيل انظر <a href="http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp" target="_blank">http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp</a> ). التعويض يسمح للتداخل الطيفية التمييز بين fluorophores. التدفق الخلوي كما يتيح التمييز من الخلايا الميتة، عموما عن طريق propidium تلطيخ يوديد. تقنية # 4. التصوير خلية واحدة المجهري هو طريقة أخرى شائعة لدراسة سلوك خلية واحدة، بل هي مناسبة تماما للدراسات التي تعتمد على الوقت وأنه يوفر أيضا القرار المكانية. والعيب الرئيسي هو أن تحليل الإنتاجية العالية ليست سوى في بداياتها في الوقت الحاضر <sتصل&gt; 9.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ham's F-10</td>
 <td>Sigma-Aldrich</td>
 <td>N-6013</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A-7906</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride Dihydrate approx. 99%</td>
 <td>Sigma-Aldrich</td>
 <td>C-3881</td>
 <td></td>
 </tr>
 <tr>
 <td>Makler Counting Chamber</td>
 <td>SEFI Medical Insruments LTD</td>
 <td>SEF-MAKL</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-3 AM</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>20 vials/50 &mu;g each</td>
 </tr>
 <tr>
 <td>Ionomycin</td>
 <td>Alomone</td>
 <td>I-700</td>
 <td></td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>Sigma-Aldrich</td>
 <td>P0130</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>S-9888</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>P-3911</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>JT Baker</td>
 <td>3506</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Magnesium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-2670</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Calcium chloride anhydrous</td>
 <td>Sigma-Aldrich</td>
 <td>C-1016</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H-3125</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>D-Glucose</td>
 <td>JT Baker</td>
 <td>1906-01</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium pyruvate</td>
 <td>Sigma-Aldrich</td>
 <td>P-2256</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium L-lactate (aprox. 99%)</td>
 <td>Sigma-Aldrich</td>
 <td>L- 7022</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Propidium Iodide </td>
 <td>Invitrogen</td>
 <td>L-7011 </td>
 <td>Component B</td>
 </tr>
 <tr>
 <td>Triton X-100 (t-Octylphenoxypolyethoxyethanol)</td>
 <td>Sigma- Aldrich</td>
 <td>X-100</td>
 <td>2.4 mM solution in water </td>
 </tr>
 <tr>
 <td>Round coverslip </td>
 <td>VWR</td>
 <td>48380 080</td>
 <td>25 mm diameter</td>
 </tr>
 <tr>
 <td>Poly-L-lysine solution </td>
 <td>Sigma-Aldrich</td>
 <td>P8920</td>
 <td></td>
 </tr>
 <tr>
 <td>Manganese chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-3634</td>
 <td></td>
 </tr>
 <tr>
 <td>Attofluor; Cell Chamber, for microscopy</td>
 <td>Life technologies</td>
 <td>A-7816</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>5x5 ml</td>
 </tr>
 </tbody>
</table>

measuring intracellular ca2+ changes in human sperm using four techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry and single cell imaging

الحيوانات المنوية هي خلايا التناسلية للذكور صمم خصيصا للوصول والتعرف عليها وتندمج مع البيضة. لأداء هذه المهام، الخلايا المنوية يجب أن يكون مستعدا لمواجهة بيئة متغيرة باستمرار والتغلب على العديد من الحواجز المادية. يجري في جوهر transcriptionally وtranslationally صامتة، وهذه الخلايا متحركة تعتمد بشكل كبير على آليات متنوعة مما يشير إلى توجيه نفسها والسباحة بطريقة موجهة، ويتعامل مع ظروف بيئية قاسية خلال رحلتهم للبحث عن البيض. على وجه الخصوص، كا 2 + إشارات بوساطة أمر حيوي لعدة وظائف الحيوانات المنوية: تفعيل الحركة، وتمكين الطاقات (عملية معقدة التي تعد الحيوانات المنوية للتفاعل الجسيم الطرفي) وردود الفعل الجسيم الطرفي (حدثا exocytotic التي تسمح الانصهار الحيوانات المنوية البيضة). استخدام الأصباغ الفلورية لتتبع تقلبات الخلايا من أيون هذا له أهمية ملحوظة نظرا لسهولة تطبيقها، والحساسية، وبراعة ديتection. باستخدام بروتوكول واحد واحد صبغ التحميل نستخدم أربع تقنيات مختلفة لمراقبة فلوروميتريك الحيوانات المنوية كا 2 + ديناميات. يوفر كل تقنية المعلومات المتميزة التي تمكن القرار المكانية و / أو الزماني، وتوليد البيانات سواء في خلية واحدة ومستويات السكان الخلية.

<img alt="الشكل 1" fo:content-width="6in" fo:src="/files/ftp_upload/50344/50344fig1highres.jpg" src="/files/ftp_upload/50344/50344fig1.jpg" /> الشكل 1. ويوضح الرسم التخطيطي للبروتوكول تجريبي لعينة الحيوانات المنوية من خلال طريقة إعداد حمام السباحة. الخطوات الرئيسية لفصل الحيوانات الم?...

Watch this Scientific Journal Video about Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell 

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

الكالسيوم داخل الخلايا<sup&gt; 2 +</sup&gt; ديناميات هي مهمة جدا في علم وظائف الأعضاء الحيوانات المنوية والكالسيوم<sup&gt; 2 +</sup&gt; حساسة الأصباغ الفلورية تشكل أداة مرنة لدراستها. وتستخدم التجارب السكان (قياس التألق وتوقف تدفق قياس التألق) والتجارب خلية واحدة (التدفق الخلوي والتصوير خلية واحدة) لتعقب المكانية والزمانية [كا<sup&gt; 2 +</sup&gt;] تغيرات في خلايا الحيوانات المنوية البشرية.

قياس الكالسيوم داخل الخلايا<sup&gt; 2 +</sup&gt; التغييرات في الحيوانات المنوية البشرية باستخدام تقنيات أربعة: قياس التألق التقليدية، وتوقفت قياس التألق التدفق، التدفق الخلوي والتصوير خلية واحدة

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

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Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

20.1K Views.  Instituto de Biotecnología-Universidad Nacional Autónoma de México. الكالسيوم داخل الخلايا 2 + ديناميات هي مهمة جدا في علم وظائف الأعضاء الحيوانات المنوية والكالسيوم 2 + حساسة الأصباغ الفلورية تشكل أداة مرنة لدراستها. وتستخدم التجارب السكان (قياس التألق وتوقف تدفق قياس التألق) والتجارب خلية واحدة (التدفق الخلوي...

Video: Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

تقنيات التصوير كاليفورنيا 2 + في اشارة الى الحيوانات المنوية البشرية

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Flow Cytometry Purification of Mouse Meiotic Cells

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 unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

تدفق تنقية الخلوي للخلايا الفأر الانتصافي

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 ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

قياس حركية تقدم دورة الخلية مع وصفها الأيض والتدفق الخلوي

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

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.

الأمثل أساليب النمذجة وتلوين لرصد انتشار استخدام الأصباغ انقسام الخلية خلية تتبع

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

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

ال<em&gt; القيطم</em&gt; سيت قص مفتوحة الفازلين جاب الجهد المشبك تقنية مع التألق

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

تقنيات لتحليل خارج الخلية الحويصلات عن طريق التدفق الخلوي

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

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.

تحليل معلقات خلية معزولة من الأنسجة الصلبة من قبل الطيفي التدفق الخلوي

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 ...

Assessing Autophagic Flux by Measuring LC3, p62, and LAMP1 Co-localization Using Multispectral Imaging Flow Cytometry

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via the lysosome and are recycled. During autophagy, cytoplasmic LC3 protein is lipidated and recruited to the autophagosomal membranes. The autophagosome then fuses with the lysosome to form the autolysosome, where the breakdown of the autophagosome vesicle and its contents occurs. The ubiquitin-associated protein p62, which binds to LC3, is also used to monitor autophagic flux. Cells undergoing autophagy should demonstrate the co-localization of p62, LC3, and lysosomal markers. Immunofluorescence microscopy has been used to visually identify LC3 puncta, p62, and/or lysosomes on a per-cell basis. However, an objective and statistically rigorous assessment can be difficult to obtain. To overcome these problems, multispectral imaging flow cytometry was used along with an analytical feature that compares the bright detail images from three autophagy markers (LC3, p62 and lysosomal LAMP1) and quantifies their co-localization, in combination with LC3 spot counting to measure autophagy in an objective, quantitative, and statistically robust manner.

Here, multispectral imaging flow cytometry with an analytical feature that compares bright detail images of 3 autophagy markers and quantifies their co-localization, along with LC3 spot counting, was used to measure autophagy in an objective, quantitative, and statistically robust manner.

تقييم التمويه الذاتي عن طريق قياس LC3، p62، و LAMP1 المشاركة في التعريب باستخدام قياس التدفق المتعدد الأطياف الخلوي

Autophagy is a catabolic pathway in which normal or dysfunctional cellular components that accumulate during growth and differentiation are degraded via ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

قياس اندوريدوبليكيشن بقياس التدفق الدرنات المعزولة بروتوبلاستس

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

تطبيقات رسم الخرائط الزمانية وتقنيات تحليل الجسيمات للتحديد الكمي لداخل الخلية Ca2 + إشارات في الموقع

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...