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الجزء 1. إعداد المتوسطة صلبة لنمو النباتات. نحن عادة ما تتركز على النباتات الثقافة المتوسطة (MS) وسكوغ Murasige تستكمل مع السكروز 1 ٪ و 0.7 ٪ أجار. فهو يحتوي على كل العناصر الضرورية للحفاظ على النباتات السليمة. لإعداد MS 1L من وسائل الاعلام الرقم الهيدروجيني 5.7 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 2،15 غرام من مسحوق MS في 800 مليلتر من الماء في زجاجة 1.5 2.0L autoclavable. </li><li style=";text-align:right;direction:rtl"> وضع شريط التحريك في زجاجة ومسحوق يذوب MS خلال إثارة المتوسطة على طبق من التحريك. </li><li style=";text-align:right;direction:rtl"> مع التحريك ، إضافة ، قطرة قطرة ، 1N KOH لضبط درجة الحموضة من المتوسط ​​إلى 5.7 MS. </li><li style=";text-align:right;direction:rtl"> إضافة 10 غرام من السكروز ، والحفاظ على التحريك. </li><li style=";text-align:right;direction:rtl"> إضافة 7 غرام من آغار *. </li><li style=";text-align:right;direction:rtl"> ضبط الحجم النهائي للمتوسط ​​على استعداد ل1000 مل وتعقيم التعقيم بواسطة.* </li><li style=";text-align:right;direction:rtl"> مكان تعقيمها المتوسطة على طبق من التحريك وتبريده مع التحريك ، بحيث آغار ولن يعجل في الجزء السفلي من القمقم. يبرد المتوسط ​​إلى 60 درجة مئوية. ~ * </li><li style=";text-align:right;direction:rtl"> صب 100 ملم لوحات بتري. لوحات في الحفاظ على 4 درجات مئوية. </li></ol> * نصيحة 1 : لا تحاول حل آغار. فإنه solubilize خلال التعقيم. * نصيحة 2 : حافظ على شريط التحريك في زجاجة خلال التعقيم ، ستحتاج في وقت لاحق. * نصيحة 3 : إذا كنت يمكن أن تبقي يديك على الزجاجة تعقيمها لمدة 10 ثانية ، فهذا يعني أن متوسط ​​هدأت بما فيه الكفاية ، ويمكنك البدء في صب لوحات. الجزء 2. المواد النباتية وشروط النمو. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تعقيم البذور من thaliana Arabidopsis <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان 100 ملغ من البذور في أنابيب microcentrifuge إيبندورف وإضافة 1 مل من الايثانول 70 ٪. مزيج جيد واحتضان لمدة 2 دقيقة. تدور بذور أسفل ، ونضح طاف. </li><li style=";text-align:right;direction:rtl"> إضافة 1 مل من محلول التبييض التي تحتوي على 1.8 ٪ * 0.1 ٪ توين - 20. المزيج لمدة 10 دقيقة. تدور باستمرار البذور ونضح محلول التبييض. </li><li style=";text-align:right;direction:rtl"> هذه الخطوة لابد من القيام به في تدفق الصفحي هود. إضافة 1 مل من الماء المعقم على البذور وتخلط جيدا. تدور باستمرار البذور ، ونضح الماء. كرر هذه الخطوة 5 مرات. </li></ol></li><li style=";text-align:right;direction:rtl"> انتشار البذور على استعداد للفي الخطوة 1 لوحات (100 بذور / لوحة). </li><li style=";text-align:right;direction:rtl"> لوحات للحفاظ على المصنف 24-48 ساعة عند 4 درجات مئوية في الظلام لمدة الطبقية. </li><li style=";text-align:right;direction:rtl"> تنمو النباتات لمدة 14 يوما في غرفة النمو مع 8 ح ح light/16 الإضاءة الداكنة (على كثافة تدفق الفوتون الضوئي من 250 μmol ق م -2 -1) في 23 / ​​19 ° C درجة حرارة النظام الضوء / الظلام والنسبية 75 ٪ الرطوبة. </li></ol> * نصيحة 1 : يتم متابعة حل عن طريق تخفيف بليتش a كلوروكس المنزلية ، والتي تحتوي على 6 ٪ هيبوكلوريت الصوديوم واضاف توين - 20 إلى تركيز النهائي من 0.1 ٪. الجزء 3. عزلة protoplasts من الشتلات Arabidopsis. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 2 غرام من شريحة لمدة 14 يوما من العمر الشتلات بشفرة حلاقة جديدة في 15 مل من محلول تعقيم فلتر TVL. نحن ختم المواد النباتية في أطباق بتري معقمة المتاح. </li><li style=";text-align:right;direction:rtl"> نقل الأنسجة المقطعة في الدورق 200 مل ، أضف 20 مل من محلول أنزيم فلتر تعقيم ، دوامة الدورق السماح الأنسجة الاختلاط مع محلول أنزيم ، مع تغطية parafilm ورقائق الألومنيوم. </li><li style=";text-align:right;direction:rtl"> اهتزاز الأنسجة النباتية في 35 دورة في الدقيقة في الظلام في درجة حرارة الغرفة ل16-18 ساعة. </li><li style=";text-align:right;direction:rtl"> جمع protoplasts صدر في 50 مل أنبوب فالكون بواسطة النخل من خلال 8 طبقات من القماش والجبن ، وقبل الرطب في W5 الحل. </li><li style=";text-align:right;direction:rtl"> protoplasts غربال من مرة واحدة أكثر الجبن القماش بواسطة غسل القماش مع 15 مل من محلول W5. </li><li style=";text-align:right;direction:rtl"> بعناية protoplasts تراكب مع 10 مل من محلول W5 ، لا تعكر صفو التدرج السكر ، لمدة 7 دقائق بنسبة 100 غرام الطرد المركزي </li><li style=";text-align:right;direction:rtl"> جمع 10 مل من protoplasts في واجهة من محلول أنزيم W5 والحل (الشكل 1B) ونقل إلى 50 عضوا جديدا أنبوب فالكون مل. </li><li style=";text-align:right;direction:rtl"> إضافة 15 مل W5 الحلول الطرد المركزي لمدة 5 دقائق عند 60 ز إزالة طاف. </li><li style=";text-align:right;direction:rtl"> غسل protoplasts خالية من محلول أنزيم عن طريق إعادة التعليق في protoplasts 15ml W5 من أجهزة الطرد المركزي ، محلول لل5min عند 60 ز </li><li style=";text-align:right;direction:rtl"> إزالة ، resuspend طاف protoplasts مكعبات في الحل W5 1 - 3ml. </li><li style=";text-align:right;direction:rtl"> تقييم العائد الجبلة المجردة من قبل خلية العد مع عدادة الكريات. </li></ol> الجزء 4. الكواشف : TVL : 0.3 M السوربيتول و 50 ملي CaCL 2 - تعقيم وتصفية المحل في -20 درجة مئوية. الحل انزيم : 0.5 M السكروز ، 10 ملي MES - ​​KOH [الرقم الهيدروجيني 5،7] ، 20 ملي CaCl 2 ، 40 ملم بوكل ، سلولاز 1 ٪ (Onozuka R - 10) ، Macerozyme 1 ٪ (R10). فلتر تعقيم واستخدام حديثا. W5 الحل : 0.1 ٪ (W / V) الجلوكوز ، 0.08 ٪ (W / V) بوكل ، 0.9 ٪ (W / V) كلوريد الصوديوم ، 1.84 ٪ (W / V) CaCl 2 ، 2 مم MES - ​​KOH الرقم الهيدروجيني 5.7. فلتر تعقيم وتخزين في درجة حرارة الغرفة. الجزء 5 : نتائج الممثل : به الظروف Arabidopsis الثقافة وصفها في أجزاء 1 و 2 يجعل الشتلات صحية ، ومناسبة لعزل protoplasts (الشكل 1A). الجبلة المجردة التي يتم تنقيتها السكروز كثافة غراديالطرد المركزي والأنف والحنجرة وجمعها في واجهة W5 والحلول انزيم (الشكل 1B). الجبلة المجردة تستخدم إجراء العزل وصفها في الجزء 3 ونحن الحصاد عادة 05-10 أكتوبر 6 من protoplasts سليمة (الشكل 1C) من غرام واحد من شتلات جديدة. <img src="/files/ftp_upload/1149/Fig1.jpg" alt="الشكل 1" /> الشكل 1. عزلة protoplasts من الشتلات Arabidopsis. وكان تم جمعها من الأنسجة لمدة 14 يوما من العمر شتلة من Arabidopsis وتحويلها إلى protoplasts بواسطة إجراء تعديل من (تشن وHalkier ، 2000). باء ، المنقى Protoplasts بواسطة الطرد المركزي المتدرج الكثافة السكروز وجمعها في واجهة من والحل انزيم W5 uffer (السهام البيضاء). C ، برايت الحقل المجهري لprotoplasts. جمعت Microphotographs باستخدام كاميرا CCD تبريد ربطه مع Axioscope زايس 2 زائد المجهر.

<ol>
	<li>Cocking, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+for+the+Isolation+of+Plant+Protoplasts+and+Vacuoles.">A Method for the Isolation of Plant Protoplasts and Vacuoles.</a> Nature. 187, 962-963 (1960).</li><li>Chen, S., Halkier, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+glucosinolate+uptake+by+leaf+protoplasts+of+Brassica+napus.">Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.</a> J Biol Chem. 275, 22955-22960 (2000).</li><li>Robert, S., Zouhar, J., Carter, C., Raikhel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+vacuoles+from+Arabidopsis+rosette+leaf-derived+protoplasts.">Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts.</a> Nat. Protocols. 2, 259-262 (2007).</li><li>Yoo, S. D., Cho, Y. H., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+mesophyll+protoplasts:+a+versatile+cell+system+for+transient+gene+expression+analysis.">Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis.</a> Nat Protoc. 2, 1565-1572 (2007).</li><li>Zhai, Z., Sooksa-nguan, T., Vatamaniuk, O. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+RNAi+as+a+reverse+genetic+approach+for+gene+functional+analysis+in+protoplasts.">Establishing RNAi as a reverse genetic approach for gene functional analysis in protoplasts.</a> Plant Phys. 149, 642-652 (2009).</li></ol>

وقد قدم براءة اختراع مؤقتة واصفا هذا الإجراء لاستخدامها في التحليلات الفنية للجينات النباتية مع مركز كورنيل للمؤسسات التكنولوجيا وتسويقها (CCTEC) ، حزيران 2008. المفكرة لا 50341/015001 : Vatamaniuk ، موافق ، وتشاي ، Z. التحليلات الفنية للأسلوب السريع للجينات النباتية.

لضمان عالية الغلة من الجبلة المجردة سليمة من المهم جدا البدء مع النباتات صحية. استخدام فلتر تعقيم حلول لعزل protoplasts. تذكر أن protoplasts هشة. لذلك ، عندما كنت التعامل مع protoplasts ، لا يختلطان الماصة أو دوامة منهم منذ ذلك بقوة ستحد منها. بدلا من ذلك ، مزيج protoplasts عن طريق تناوب ببط?...

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<td>Murashige and Skoog Basal Salt Mixture (MS)</td>
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<td>M5524</td>
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<td>Agar</td>
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isolation of protoplasts from tissues of 14-day-old seedlings of arabidopsis thaliana

Protoplasts هي الخلايا النباتية التي كانت جدران الزنزانة لازالة إنزيمي. وكانت أول من العزلة protoplasts من الأنسجة النباتية المختلفة أكثر من 40 عاما<sup&gt; 1</sup&gt; ومنذ ذلك الحين تم تكييفها لدراسة مجموعة متنوعة من العمليات الخلوية ، مثل توطين التحت خلوية من البروتينات ، والعزلة من عضيات سليمة واستهدفت تعطيل الجينات التي تدخل RNA المزدوج تقطعت بهم السبل (رني)<sup&gt; 2-5</sup&gt;. معظم بروتوكولات العزل الجبلة المجردة استخدام أنسجة نبات من Arabidopsis الناضجة (مثل 35 يوما من العمر النباتات)<sup&gt; 2-4</sup&gt;. نحن تعديل البروتوكولات القائمة من خلال توظيف Arabidopsis الشتلات لمدة 14 يوما من عمره. في هذا الإجراء ، أسفرت عن 14 غراما واحدا من أيام عمره الشتلات 5 10<sup&gt; 6</sup&gt; -10<sup&gt; 7</sup&gt; protoplasts التي لا تزال على حالها لا يقل عن 96 ساعة. العائد من protoplasts من الشتلات هو مقارنة مع الاستعدادات من أوراق من Arabidopsis ناضجة ، ولكن بدلا من 35-36 يوما ، يتم الانتهاء من العزلة protoplasts في 15 يوما. وهذا يسمح بخفض الوقت ونمو مساحة الغرفة التي مطلوبة من أجل عزل protoplasts عندما تستخدم محطات ناضجة ، ويعجل من الدراسات اللاحقة التي تتطلب protoplasts سليمة.

Watch this Scientific Journal Video about Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana at JoVE.com

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

هذا الفيديو يظهر إجراء لعزل protoplasts سليمة من أنسجة لمدة 14 يوما من العمر شتلة من Arabidopsis. بالنظر إلى أن protoplasts معزولة لا تزال سليمة على الأقل 96h ومعزولة من الشتلات بدلا من شهر واحد من العمر محطات ناضجة ، وهذا الإجراء يعجل المقايسات التي تتطلب protoplasts سليمة.

عزلة Protoplasts من أنسجة لمدة 14 يوما من عمره الشتلات thaliana Arabidopsis</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

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Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

35.9K Views.  Cornell University. هذا الفيديو يظهر إجراء لعزل protoplasts سليمة من أنسجة لمدة 14 يوما من العمر شتلة من Arabidopsis. بالنظر إلى أن protoplasts معزولة لا تزال سليمة على الأقل 96h ومعزولة من الشتلات بدلا من شهر واحد من العمر محطات ناضجة ، وهذا الإجراء يعجل المقايسات التي تتطلب protoplasts سليمة.

Video: Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

مضان الفرز الخلية النباتية المنشط من Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

الأزهار وتراجع تحويل thaliana Arabidopsis لتفحص pTSO2 : β غلوكورونيداز التعبير مراسل جين

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Arabidopsis thaliana التنميط Glycerolipid القطبية التي اللوني طبقة رقيقة (TLC) اقترن اللوني الغاز السائل (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

عزل الخلايا الليفية عادي والسرطان يرتبط بها من الأنسجة الطازجة من الإسفار خلية المنشط الفرز (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

العزلة الفعالة والسريعة من الأجنة المبكرة مرحلة من<em&gt; thaliana نبات الأرابيدوبسيس</em&gt; بذور

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

الأيضية وصفها وغشاء التقطير للالمقارن تحليل البروتين من<em&gt; نبات الأرابيدوبسيس thaliana</em&gt; الثقافات تعليق خلية

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

تلطيخ النسيجية من<em&gt; نبات الأرابيدوبسيس thaliana</em&gt; الخليوي الثانوية ستريت عناصر

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

عزل القائم على تقارب من معلم النواة من<em&gt; ذبابة الفاكهة</em&gt; الأنسجة لتحليل التعبير الجيني

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

تحليل سريع من الظواهر الإيقاعية في نبات الأرابيدوبسيس جبلة مجردة Transfected مع ساعة مراسل الفلورسنت

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light conditions, which are preferably variable in quantity and quality so that studies under different setups can be conducted. These requirements are met by climatic chambers featuring light emitting diodes (LED) lights, which can &#8211; in contrast to fluorescent lights &#8211; be set to different wavelengths. LEDs are energy conserving and emit virtually no heat even at light intensities, which often constitutes a problem with other light sources. The presented protocol provides a step-by-step guidance of how to program a climatic chamber equipped with variable LED lights as well as describing several approaches for in depth analysis of growth phenotypes. Depending on the experimental set-up various characteristics of the growing plants can be observed and analyzed. Here we describe how to determine fresh weight, leaf area, photosynthetic activity, and stomatal density. We demonstrate that in order to obtain reliable data and draw valid conclusions it is mandatory to use a sufficient number of individuals for statistical evaluation. Taking too few plants for this kind of analysis results in high statistical errors and consequently in less clear interpretations of the data.

Analysis of Arabidopsis thaliana Growth Behavior in Different Light Qualities

Here, we present a protocol for studying plant growth behavior and especially phenotypes in a reproducible manner. We show how to provide variable and at the same time stable light conditions. Proper analyses depend on sufficient sample numbers and valid statistical evaluations.

تحليل "سلوك نمو" نبات التمويل في "نوعيات مختلفة من الضوء"

Plant biologists often need to observe the growth behavior of their chosen species. To this end, the plants need constant environmental and stable light ...