فإن الكتاب أود أن أشكر لورانس كوبر ، Harjeet سينغ ، وHurton ينكا لعملهم في خلق K562 aAPC الأولية وناقلات mIL21 الانصهار. تم توفير التمويل لهذا العمل من قبل برنامج UT أندرسون عالم طبيب ، مؤسسة Baldrick سانت ، وأساطير Friendswood.

<ol>
	<li>McKenna, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+manufacturing+practices+production+of+natural+killer+cells+for+immunotherapy:+a+six-year+single-institution+experience.">Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience.</a> Transfusion. 47, 520-520 (2007).</li><li>Koehl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+highly+purified+NK+cells+for+immunotherapy+after+haploidentical+stem+cell+transplantation+in+children.">ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.</a> Klinische Padiatrie. 217, 345-345 (2005).</li><li>Klingemann, H. G., Martinson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+natural+killer+cells+for+clinical+applications.">ex vivo expansion of natural killer cells for clinical applications.</a> Cytotherapy. 6, 15-15 (2004).</li><li>Ayello, J. <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+cord+blood+natural+killer+and+lymphokine+activated+killer+lymphocytes+following+ex+vivo+cellular+engineering.">Characterization of cord blood natural killer and lymphokine activated killer lymphocytes following ex vivo cellular engineering.</a> Biol Blood Marrow Transplant. 12, 608-608 (2006).</li><li>Carlens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+in+vitro+expansion+of+cytotoxic+human+CD3-CD56++natural+killer+cells.">A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells.</a> Hum Immunol. 62, 1092-1092 (2001).</li><li>Boissel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Umbilical+cord+mesenchymal+stem+cells+increase+expansion+of+cord+blood+natural+killer+cells.">Umbilical cord mesenchymal stem cells increase expansion of cord blood natural killer cells.</a> Biol Blood Marrow Transplant. 14, 1031-1031 (2008).</li><li>North, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-primed+human+natural+killer+cells+lyse+NK-resistant+tumor+targets:+evidence+of+a+two-stage+process+in+resting+NK+cell+activation.">Tumor-primed human natural killer cells lyse NK-resistant tumor targets: evidence of a two-stage process in resting NK cell activation.</a> J Immunol. 178, 85-85 (2007).</li><li>Berg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical-grade+ex+vivo-expanded+human+natural+killer+cells+up-regulate+activating+receptors+and+death+receptor+ligands+and+have+enhanced+cytolytic+activity+against+tumor+cells.">Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells.</a> Cytotherapy. 11, 341-341 (2009).</li><li>Fujisaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+highly+cytotoxic+human+natural+killer+cells+for+cancer+cell+therapy.">Expansion of highly cytotoxic human natural killer cells for cancer cell therapy.</a> Cancer Res. 69, 4010-4010 (2009).</li><li>Fujisaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+potential+of+human+natural+killer+cells.">Replicative potential of human natural killer cells.</a> Br J Haematol. 145, 606-606 (2009).</li><li>Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+of+natural+killer+cells+with+high+cytotoxicity+by+K562+cells+modified+to+co-express+major+histocompatibility+complex+class+I+chain-related+protein+A,+4-1BB+ligand,+and+interleukin-15.">Ex vivo expansion of natural killer cells with high cytotoxicity by K562 cells modified to co-express major histocompatibility complex class I chain-related protein A, 4-1BB ligand, and interleukin-15.</a> Tissue Antigens. 76, 467-467 (2010).</li></ol>

الإعلان عن أي تضارب في المصالح.

NK توسيع الخلايا وتنشيط وسائل الإعلام (NKEM) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 90 ٪ RPMI 1640 (Cellgro) </li><li style=";text-align:right;direction:rtl"> 10 ٪ الجنين المصل البقري (Gibco) </li><li style=";text-align:right;direction:rtl"> 1X البنسلين / الستبرتوميسين (Cellgro) </li><li style=";text-align:right;direction:rtl"> L - 1X الجلوتامين (Gibco) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تصفية بتعقيم وسائل الاعلام قبل الاستخدام. </li></ul></li><li style=";text-align:right;direction:rtl"> 50 U / مل IL2 (Proleukin واللقاحات والتشخيص نوفارتيس ، وشركة) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> -- المخففة من الأسهم IU / ميكرولتر 200. إضافة إلى IL2 المبلغ المطلوب من وسائل الإعلام فقط قبل استخدام في كل مرة. </li></ul></li></ul> وPBMC NK زنزانة انفرادية <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Ficoll - Paque (GE للرعاية الصحية) </li><li style=";text-align:right;direction:rtl"> Alsever محلول (سيجما) </li><li style=";text-align:right;direction:rtl"> RosetteSep خلية NK الإنسان كوكتيل التخصيب (Stemcell تكنولوجيز) </li></ul> NK الفحص الخلوي السمسة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Calcein - AM (Invitrogen) <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الأسهم هو الحل في متجر ، 1mg / مل المجمدة في 50 aliquots ميكرولتر. تذوب في وسائل الاعلام لتحقيق NKEM التخفيف المطلوب لتلوين الخلية. </li></ul></li></ul> الأضداد يتم سرد قائمة من الأجسام المضادة المستخدمة في الخلية phenotyping ناغورني كاراباخ في الجدول أدناه ؛ <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td></td><td> الجسم المضاد </td><td> حجم لكل اختبار </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> أنبوب 1 </td><td valign="top"> Isotype FITC Isotype FITC Isotype FITC Isotype FITC FACS العازلة اجمالى حجم التداول </td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen </td><td valign="top"> 555748 555749 557224 340442 </td></tr><tr><td> أنبوب 2 </td><td valign="top"> CD56 FITC NKp30 PE NKp44 PE NKp46 PE CD3 PE - Cy5 CD16 اليكسا 647 FACS العازلة اجمالى حجم التداول </td><td valign="top"> 5 5 5 5 5 5 70 100 </td><td valign="top"> دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen </td><td valign="top"> 340410 558407 558563 557991 555341 557710 </td></tr><tr><td> أنبوب 3 </td><td valign="top"> CD56 FITC KIR2DL1 PE KIR2DL2 / 3 PE KIR3DL1 PE CD3 PE - Cy5 NKG2D APC FACS العازلة اجمالى حجم التداول </td><td valign="top"> 5 5 5 5 5 5 70 100 </td><td valign="top"> دينار بحريني Pharmingen R &amp; D نظم Miltenyi بيوتيك R &amp; D نظم دينار بحريني Pharmingen دينار بحريني Pharmingen </td><td valign="top"> 340410 FAB1844P 130-092-618 FAB12251P 555341 558071 </td></tr><tr><td> أنبوب 4 </td><td valign="top"> CD56 FITC CD11b PE CD3 PE - Cy5 CD27 APC FACS العازلة اجمالى حجم التداول </td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen </td><td valign="top"> 340410 555388 555341 558664 </td></tr><tr><td> أنبوب 5 </td><td valign="top"> CD56 FITC CD266 (DNAM - 1) PE CD3 PE - Cy5 CD160 Alexa647 FACS العازلة اجمالى حجم التداول </td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen eBiosciences </td><td valign="top"> 340410 559789 555341 51-1609-42 </td></tr><tr><td> أنبوب 6 </td><td valign="top"> CD56 FITC CD244 (2B4) PE CD3 PE - Cy5 CD197 (CCR7) APC FACS العازلة اجمالى حجم التداول </td><td valign="top"> 5 5 5 5 80 100 </td><td valign="top"> دينار بحريني Pharmingen دينار بحريني Pharmingen دينار بحريني Pharmingen eBiosciences </td><td valign="top"> 340410 550816 555341 17-1979-42 </td></tr></tbody></table>

expansion, purification, and functional assessment of human peripheral blood nk cells

الطبيعية القاتلة (ناغورني كاراباخ) الخلايا تلعب دورا هاما في مراقبة المناعة ضد مجموعة متنوعة من الكائنات الحية الدقيقة المعدية والأورام. قيدت توافر محدود من الخلايا القاتلة الطبيعية والقدرة على التوسع في تطوير المختبر المناعي خلايا NK. نحن هنا تصف طريقة لتوسيع بكفاءة كميات هائلة من وظيفية NK فيفو السابقين الخلايا باستخدام الخلايا K562 معربا عن غشاء محدد IL21 ، باعتبارها خلية مستضد تقديم الاصطناعي (aAPC). وقد استخدمت العلاج بالخلايا NK بالتبني حتى الآن منتج الخلية التي حصلت عليها الدولة المستقرة leukapheresis من المانحين تليها استنفاد الخلايا التائية أو التحديد الإيجابي للخلايا NK. وعادة ما يتم تنشيط المنتج في IL - 2 ثم بين عشية وضحاها وتدار في اليوم التالي 1. بسبب التردد المنخفض من الخلايا القاتلة الطبيعية في الدم المحيطي ، وقد تم تسليم عدد قليل نسبيا من الخلايا القاتلة الطبيعية في التجارب السريرية. وقد عجز لنشر الخلايا القاتلة الطبيعية في المختبر العامل المحدد لتوليد أرقام خلية كافية لنتائج سريرية الأمثل. التوسع في بعض الخلايا القاتلة الطبيعية (5-10 أضعاف على مدى 1-2 أسابيع) ويمكن تحقيقه من خلال وحده IL - 2 جرعة عالية 2. ويمكن تنشيط خلايا تي ذاتي التوسط NK توسيع الخلية ، ويفترض أيضا من خلال الافراج عن خلوى المحلية 3. يمكن أن تدعم سدى مع الوسيطة أو الخلايا الاصطناعية تقديم المستضد (aAPCs) دعم التوسع في الخلايا القاتلة الطبيعية سواء من الدم المحيطي ودم الحبل السري 4. يجري تسويق NKp46 المشتركة وتفعيل CD2 بواسطة الأجسام المضادة المغلفة الخرز للتوسع الخلايا القاتلة الطبيعية (Miltenyi بيوتيك ، صحراء كاليفورنيا) ، مما أدى إلى التوسع في ما يقرب من 100 مرات في 21 يوما. التجارب السريرية باستخدام aAPC الموسع أو تنشيط خلايا NK جارية ، واحد باستخدام خط خلية اللوكيميا CTV - 1 لرئيس الوزراء وتنشيط خلايا NK 5 دون توسع كبير. والمحاكمة الثانية تستخدم LCL - EBV للتوسع الخلية ناغورني كاراباخ ، وتحقيق التوسع يعني 490 مرات في 21 يوما 6. ثالث يستخدم لaAPC K562 المستندة transduced مع 1BBL - 4 (CD137L) و 7 غشاء محدد IL - 15 (MIL - 15) ، والتي حققت توسعا يعني NK 277 مرات في 21 يوما. على الرغم من أن الخلايا القاتلة الطبيعية باستخدام موسع - K562 - 41BBL mIL15 aAPC السامة للخلايا هي غاية في المختبر والمجراة مقارنة مع الخلايا القاتلة الطبيعية unexpanded ، والمشاركة في ADCC ، يقتصر انتشارها الشيخوخة التي نسبت الى تقصير التيلومير 8. وأفيد في الآونة الأخيرة توسعا 350 ضعفا من الخلايا القاتلة الطبيعية باستخدام K562 ميكا الإعراب ، و 4 و 9 - 1BBL IL15. وصف أسلوبنا في توسيع الخلايا القاتلة الطبيعية تنتج هنا الانتشار السريع لخلايا NK الشيخوخة دون تحقيق التوسع وسيطة 21000 مرات في 21 يوما.

Watch this Scientific Journal Video about Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells at JoVE.com

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

نحن هنا تصف طريقة لتوسيع بكفاءة وتنقية أعداد كبيرة من الخلايا القاتلة الطبيعية البشرية وتقييم وظائفها.

توسيع وتنقية ، والتقييم الوظيفي للخلايا الدم المحيطية الإنسان NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

expansion-purification-functional-assessment-human-peripheral-blood

Research

JoVE Journal

Immunology and Infection

48.1K Views.  MD Anderson Cancer Center - University of Texas. نحن هنا تصف طريقة لتوسيع بكفاءة وتنقية أعداد كبيرة من الخلايا القاتلة الطبيعية البشرية وتقييم وظائفها.

Video: Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

تنقية سكان خلية معينة من قبل الإسفار الفرز الخلية المنشط (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

التوسع في الدم المحيطي الإنسان خلايا T γδ باستخدام Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

محمي مستضدات خلايا الدم الحمراء وظيفية بواسطة الغشاء مسمار Polyglycerols Hyperbranched الاتفاق

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

كشف الجليكوجين في خلايا الدم المحيطي وحيدات النوى مع الدوري حمض شيف تلطيخ

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

توليد خلايا T الإنسان مستضد خيفي محددة من الدم المحيطي

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

نهج مؤتمتة شبه لإعداد الجسم المضاد الكوكتيلات لتحليل Immunophenotypic في الدم المحيطي الإنسان

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

ديكستران يعزز كفاءة توصيل لينتيفيرال الخلايا ناغورني كاراباخ الابتدائي مورين والبشرية

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

تحليل القائم على الخلوي سيتوتوكسيسيتي تدفق لتقييم نشاط الخلايا البشرية ناغورني-كاراباخ

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

توسيع وتنقية ، والتقييم الوظيفي للخلايا الدم المحيطية الإنسان NK

PLAYLIST