이 연구는 캐나다 혈액 서비스 (CBS)과 보건 과학 (CIHR) 연구 파트너십 기금의 캐나다 연구소에 의해 자금을 지원했다. 저자는 자신의 연구 시설의 사용에 대한 혈액 연구 UBC 센터에서 LMB Macromolecule 허브 감사드립니다. 인프라 시설 혁신에 대한 캐나다 재단 (CFI)과 건강 연구에 대한 마이클 스미스 재단 (MSFHR)에 의해 지원됩니다. R. Chapanian은 (CIHR / CBS) 박사 수혈 과학 장학금 및 MSFHR 연구 연수 후 박사 교제의 수신자의 수상자이다. JN Kizhakkedathu는 MSFHR 경력 탐정 학술 상 수상자이다.

A. Hyperbranched Polyglycerol의 변경 (SS-HPG) <ol><li> 장소 둥근 바닥 플라스크에 60 kDa (0.5 g, 0.0083 mmol)을 HPG 동결 건조 및 90 진공 박을 건조 ° C. </li><li> 실내 온도에 휴대용 술병을 냉장하고, 무수 피리딘 (3 ML)에서 건조 HPG를 해산. </li><li> 카르 복실 그룹과 HPG에 약 여덟 수산기 그룹을 functionalize하려면 HPG 솔루션으로 dimethylaminopyridine의 촉매 금액 (피리딘 5 MG / ML 솔루션의 한 방울)을 추가합니다. 이 혼합물에 10 분 이상 현명한 0.5 ML 피리딘 드롭에 용해 (0.0067 g, 0.0664 mmol), succinic 무수물을 추가합니다. 아르곤에 따라 실온에서 하루 아침에 혼합물을 저어. </li><li> 50 ML의 원심 분리기 튜브의 차가운 아세톤 (4 ° C)의 40 ML에있는 혼합물을 침전, 15 분 동안 27,000 XG에서 Beckman J2-MC의 원심 분리기를 사용하여 원심 분리기. 표면에 뜨는을 가만히 따르다하고, 실온에서 아르곤과 함께 플러싱에 의해 잔류 아세톤을 제거합니다. </li><li> 카르복시을 활성화하려면succinimidyl 신산 (SS) 단체와 난 그룹은, 무수 DMF 3 ML에있는 카르 복실-작용 HPG를 해산. HPG 솔루션에 N-hydroxysuccinimide (0.0077 g, 0.0664 mmol) 및 N, N&#39;-diisopropylcarbodiimide을 (0.0084 g, 0.0664 mmol)을 추가 및 아르곤 이하 실온에서 하루 아침에 혼합물을 저어. </li><li> 차가운 아세톤의 강수량에 의해 SS-HPG를 정화. </li><li> 아르곤과 함께 플러싱에 의해 잔류 아세톤을 제거합니다. </li><li> NMR 분석 - 수정 HPG의 순도와 양자 (1 H)의 카르 복실와 SS 작용의 정도를 결정합니다. </li></ol> B. 전체 혈액 수집 및 적혈구의 분리 (적혈구) <ol><li> 구연산의 vacutainer 관으로 건강한 인간 기증자를 동의에서 전체 혈액 (40 % 혈소판, 3 ML)를 수집합니다. </li><li> 4 분 1,000 XG에 시트르산 튜브를 원심 분리기. </li><li> 플라즈마와 혈소판을 포함하는 표면에 뜨는을 제거하고, 백혈구와 platele를 포함하는 중간 버피 코트파스퇴르 피펫을 사용하여 TS. </li><li> 12 ML 팔콘의 원심 분리기 튜브에 포장 적혈구 (80 % 혈소판, 1.2 ML)를 전송하고, 적혈구를 씻어 PBS 버퍼를 (8 ML, 산도 = 8.0)를 추가합니다. </li><li> 균일 한 세포 분포를 얻기 위해 반전하여 적혈구를 섞는다. </li><li> 4 분 1,000 XG에서 매 튜브를 원심 분리기하고, 표면에 뜨는을 제거합니다. </li><li> 단계 5와 6을 반복하여 PBS 두 번으로 적혈구를 씻으십시오. </li><li> 장소 1.6 ML Eppendorf 튜브에 (100 μl) 적혈구를 포장, 20 % 혈소판의 적혈구를 얻기 위해 PBS 버퍼 (300 μl, 산도는 = 8.0)을 추가합니다. </li></ol> C. HPG는 적혈구로 이식 <ol><li> 즉시 단계 A6의 아세톤에 수정 HPG, 장소의 강수량 후 유리관 (1 DRAM) 및 PBS 버퍼 (300 μl, 산도는 = 8.0)에 용해에 SS-HPG (150 MG). </li><li> 20 % 혈소판에 SS-HPG 폴리머 솔루션을 추가하면 400 μl와 0.5 mm까지에서 3 mm까지 범위 SS-HPG 농도의 최종 볼륨을 얻기 위해 적혈구을 빨기도 했죠. 예를 들어 0.5 밀리미터로 치료 적혈구를 준비하는 SS-HPG, 세탁 RBC에서 PBS의 36 μl를 제거하고 SS-HPG 폴리머 솔루션 36 μl를 놓습니다. </li><li> 소용돌이 부드럽게 정지 1 시간 동안 실온에서 궤도 셰이커에 Eppendorf 튜브에 넣습니다. </li><li> 4 분 1,000 XG에서 Eppendorf 튜브 원심 분리기하고 표면에 뜨는을 피펫. </li><li> , 적혈구을 씻고 원심 분리기하고 표면에 뜨는을 제거하는 PBS 1 ML을 추가합니다. </li><li> 한 ML의 생리를 추가하고 배 5 단계에서 적혈구을 씻는다. </li><li> 20 % 혈소판의 최종 농도로 가득 적혈구의 100 μl (80 % 혈소판)에 생리 300 μl를 추가합니다. </li></ol> 수정일 적혈구를 HPG의 D. 특성 I.의 보수 매개의 용해 <ol><li> 건강한 사람의 기증자로부터 BD vacutainer 유리 혈청 관에 전체 혈액의 8 주변 ML를 수집하고, 30 분 동안 실온에서 응고에 혈액을 수 있습니다. </li><li> 10 분에 2,000 XG에서 튜브를 원심 분리기와 혈청 3 약 ML를 수집합니다. </li><li> seru 중 하나 밀리리터m은 열 inactivated 혈청를 준비하는 30 분에 60 ° C에서 물을 욕조에 배치되었습니다. </li><li> 20 % 혈소판 HPG 수정 적혈구 나 수정되지 않은 적혈구의 60 μl를 포함 Eppendorf 튜브에 신선한 혈청 또는 열 inactivated 혈청 60 μl를 추가합니다. </li><li> 37 1 시간에 적혈구를 품다 ° C. </li><li> 세포 현탁액에서 헤모글로빈의 양, 장소 96 잘 플레이트에 세중의에서 수정 적혈구 나 수정되지 않은 세포를 HPG의 20 % 혈소판의 5.9 μl을 수량화 할 수 있습니다. Drabkin의 시약 (spectrophotomerically 헤모글로빈의 양을 정량화하는 데 사용되는 시약)의 294 μl를 추가합니다. 및 아웃 pipetting하여 Drabkin의 시약으로 세포를 섞는다. 스펙트럼 MAX 190 판 리더를 사용하여 540 nm 정도의 헤모글로빈 cyanoderivati​​ve의 흡광도를 측정합니다. </li><li> 표면에 뜨는에서 헤모글로빈의 양을 정량화하기 위해 1 분에 13,000 XG에서 세포를 원심 분리기. 96 잘 플레이트에 세중의에 표면에 뜨는 장소 50 μl. Drabkin의 시약 250 μl를 추가합니다. Drabkin의 시약과 혼합 세포및 아웃 pipetting하여. 스펙트럼 MAX 190 판 리더를 사용하여 540 nm 정도의 헤모글로빈 cyanoderivati​​ve의 흡광도를 측정합니다. </li><li> 샘플의 표면에 뜨는에서 헤모글로빈의 비율 (7 단계) 및 총 헤모글로빈 (단계 6) 8, 11, 12에서 lysed 세포의 양을 정량화. </li></ol> II. MTS 카드를 사용하여 전공 및 부전공 항원의 위장 <ol><li> 장소 50 Eppendorf 튜브에 HPG 수정 RBC (10 % 혈소판), 1 분 1,000 XG에서 원심 분리기의 μl, 그리고 표면에 뜨는을 제거합니다. </li><li> 세포 펠렛에 MTS 희석제의 110 μl를 추가하고 및 아웃 pipetting하여 세포 현탁액을 균질화. </li><li> 각 미니 겔 항목에 세포 현탁액 11 μl를 추가합니다. 또한 동안 젤을 터치하지 마십시오. </li><li> 원심 분리기 카드 소지자에 MTS 카드를 찾아 6 분에 156 XG에 원심 분리기. </li><li> 표면 항원의 보호는 제조에 따라, 미니 젤 열에서 RBC의 위치에서 결정된다R 설명입니다. </li></ol> III. 수성 2 상 파티션 측정 <ol><li> Dextran 500 kDa 13에 따라 (5 % dextran 50 만, 4% PEG 8K, 150 MM NaCl, 10 MM 인산) 구성 / PEG 8 kDa 2 상 분리 시스템을 준비합니다. </li><li> 두 단계 시스템 (아래의 dextran)을 얻기 위해 실온에서 10 분에 433 XG에 원심 분리기. </li><li> 조심스럽게 두 단계를 분리합니다. </li><li> 표시 관 (오목 하단에 있음)에 상부 PEG 단계의 나누어지는 1.0 ML은 flicking하여 부드럽게 20 20% 혈소판 HPG 수정 RBC의 μl와 혼합 셀을 추가 할 수 있습니다. </li><li> 낮은 단계의 0.5 ML로 세포를 포함하고, flicking에 의해 부드럽게 시스템을 혼합 상단 단계의 0.5 ML를 추가합니다. </li><li> 분리하고 시스템에서 셀의 위치를​​ 결정하는 시스템 ~ 2 분 동안 벤치에 튜브를 놓습니다. 적혈구의 국산화은 (상단 PEG 단계에서, 낮은 dextran 단계에서 또는 두 가지 모두)을 수정 및 분자의 정도 기능입니다HPG의 무게. </li></ol> IV. 삼투 취약성 측정 <ol><li> 0에서 0.9 (w / V) %로 다양 농도와 NaCl 솔루션을 준비합니다. </li><li> 1.5 ML Eppendorf 튜브에 NaCl 솔루션의 장소 0.4은 ML. </li><li> NaCl 솔루션으로 적혈구 (20 % 혈소판)의 20 μl를 추가합니다. </li><li> 반전 30 분 동안 37 ° C에서 물이 욕조에 넣어하여 신중하게 믹스 세포. </li><li> 반전으로 조심스럽게 세포를 일시 중지합니다. RBC 서스펜션의 50 μl를 타고 큐벳에 배치 한 ML Drabkin의 시약,에 추가 할 수 있습니다. 및 아웃 pipetting하여 Drabkin의 시약으로 세포를 섞는다. BECKMAN 보습 바로 앞에 달린 풀 베는 날 DU 730 UV / VIS 리더를 사용하여 540 nm 정도의 헤모글로빈 cyanoderivati​​ve의 흡광도를 측정합니다. </li><li> 표면에 뜨는에서 헤모글로빈의 양을 정량화하기 위해 1 분 1,000 XG에서 세포를 원심 분리기. 큐벳에서 Drabkin의 시약 1 ML에 표면에 뜨는 200 μl를 추가합니다. 및 아웃 pipetting하여 Drabkin의 시약으로 세포를 섞는다. hemogl의 흡광도를 측정BECKMAN 보습 바로 앞에 달린 풀 베는 날 DU 730 UV / VIS 리더를 사용하여 540 nm의에서 obin cyanoderivati​​ve. </li><li> 세포 현탁액 (5 단계)에서 헤모글로빈의 총 금액에 표면에 뜨는에있는 금액 헤모글로빈의 비율 (6 단계)에서 다른 NaCl 용액에 lysed 세포의 양을 계산할 수 있습니다. </li></ol> V. 흐름 세포 계측법 측정 - 리 서스-D의 보호 (RhD)는 항원 <ol><li> 5 제어 μl 25 μl의 총 볼륨에 0.5 % BSA와 보충 PBS 버퍼로 세중의에 HPG 수정 적혈구 (20 % 혈소판)을 추가합니다. </li><li> 지수 Biodiagnostics (PA, USA)에서 구입 한 FITC 단클론 항 리 서스 D (RhD)의 25 μl를 추가합니다. </li><li> 물 욕조에 37 ° C에서 30 분의 혼합물을 품다. </li><li> 1.5 ML PBS 버퍼를 두 번으로 혼합물을 씻으십시오. 표면에 뜨는을 제거하려면 1 분 1,000 XG에 원심 분리기. </li><li> 한 ML의 생리에 적혈구를 일시 중지, 4 ML 흐름 BD의 유동 세포 계측법 관에 정지를 전송합니다. </li><li> FACSCanto II FLO를 사용하여 분석w cytometer는 RBC 제어 게이트에 5000 이벤트를 수집하고, 분석을 위해 전체 이벤트를 사용합니다. </li></ol> VI. 유동 세포 계측법 측정 - CD47의 표현 <ol><li> 1.54 제어 μl 또는 44 μl의 총 볼륨에 0.5 % BSA와 보충 PBS 버퍼로 세중의에 HPG 수정 적혈구 (20 % 혈소판)을 추가합니다. </li><li> BD Biosciences (NJ, USA)에서 구입 한 마우스 반 인간 CD47이라는 phycoerythrin (PE) 6 μl를 추가합니다. </li><li> 물 욕조에 37 ° C에서 30 분의 혼합물을 품다. </li><li> 두 배에 1.5 ML PBS 버퍼로 혼합물을 씻으십시오. 표면에 뜨는을 제거하려면 1 분 1,000 XG에 원심 분리기. </li><li> 한 ML의 생리에 적혈구를 일시 중지하고, 세포 거리며 걷게을 최소화하기 위해 26 G 8분의 5 바늘을 통과. </li><li> 4 ML 흐름 BD의 유동 세포 계측법 관에 정지를 전송합니다. </li><li> RBC 제어 게이트 만 이벤트를 인수, FACSCanto II 흐름 cytometer를 사용하여 분석합니다. </li></ol>

<ol>
	<li>Bradley, A. J., Murad, K. L., Regan, K. L., Scott, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+consequences+of+linker+chemistry+and+polymer+size+on+stealth+erythrocytes:+size+does+matter.">Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter.</a> Biochim. Biophys. Acta. 1561 (2), 147-158 (2002).</li><li>Murad, K. T., Mahany, K. L., Brugnara, C., Kuypers, F. A., Eaton, J. W., Scott, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+consequences+of+antigenic+modulation+of+red+blood+cells+with+methoxypoly(ethylene+glycol.">Structural and functional consequences of antigenic modulation of red blood cells with methoxypoly(ethylene glycol.</a> Blood. 93 (6), 2121-2127 (1999).</li><li>Scott, M. D., Murad, K. L., Koumpouras, F., Talbot, M., Eaton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+camouflage+of+antigenic+determinants:+Stealth+erythrocytes.">Chemical camouflage of antigenic determinants: Stealth erythrocytes.</a> Proc. Natl. Acad. Sci. U.S.A. 94 (14), 7566-7571 (1997).</li><li>Daniels, G., Reid, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+groups:+the+past+50+years.">Blood groups: the past 50 years.</a> Transfusion. 50 (2), 281-289 (2010).</li><li>Murad, K. L., Gosselin, E. J., Eaton, J. W., Scott, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stealth+cells:+Prevention+of+major+histocompatibility+complex+class+II-mediated+T-cell+activation+by+cell+surface+modification.">Stealth cells: Prevention of major histocompatibility complex class II-mediated T-cell activation by cell surface modification.</a> Blood. 94 (6), 2135-2141 (1999).</li><li>Kainthan, R. K., Hester, S. R., Levin, E., Devine, D. V., Brooks, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+biological+evaluation+of+high+molecular+weight+hyperbranched+polyglycerols.">In vitro biological evaluation of high molecular weight hyperbranched polyglycerols.</a> Biomaterials. 28 (31), 4581-4590 (2007).</li><li>Kainthan, R. K., Janzen, J., Levin, E., Devine, D. V., Brooks, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatibility+testing+of+branched+and+linear+polyglycidol.">Biocompatibility testing of branched and linear polyglycidol.</a> Biomacromolecules. 7 (3), 703-709 (2006).</li><li>Chapanian, R., Constantinescu, I., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+circulation,+clearance,+and+biodistribution+of+polyglycerol+grafted+functional+red+blood+cells.">In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells.</a> Biomaterials. 33 (10), 3047-3057 (2012).</li><li>Chapanian, R., Constantinescu, I., Rossi, N. A. A., Medvedev, N., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+polymer+architecture+on+antigens+Camouflage,+CD47+protection+and+complement+mediated+lysis+of+surface+grafted+red+blood+cells.">Influence of polymer architecture on antigens Camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.</a> Biomaterials. 33 (31), 7871-7883 (2012).</li><li>Rossi, N. A. A., Constantinescu, I., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+cell+surface+polymer+grafting+in+concentrated+and+nonreactive+aqueous+polymer+solutions.">Enhanced cell surface polymer grafting in concentrated and nonreactive aqueous polymer solutions.</a> J. Am. Chem. Soc. 132 (10), 3423-3430 (2010).</li><li>Rossi, N. A. A., Constantinescu, I., Kainthan, R. K., Brooks, D. E., Scott, M. D., Kizhakkedathu, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+blood+cell+membrane+grafting+of+multi-functional+hyperbranched+polyglycerols.">Red blood cell membrane grafting of multi-functional hyperbranched polyglycerols.</a> Biomaterials. 31 (14), 4167-4178 (2010).</li><li>Muzykantov, V. R., Smirnov, M. D., Domogatsky, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolytic+complement+activity+assay+in+microtitration+plates.">Hemolytic complement activity assay in microtitration plates.</a> J. App. Biochem. 7 (3), 223-227 (1985).</li><li>Walter, H., Brooks, D. E., Fisher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Partitioning in aqueous two-phase systems: theory, methods, uses, and applications to biotechnology. , (1985).</li><li>Rossi, L., Serafini, S., Pierige, F., Antonelli, A., Cerasi, A., Franternale, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte-based+drug+delivery.">Erythrocyte-based drug delivery.</a> Expert Opin. Drug Deliv. 2 (2), 311-322 (2005).</li><li>Walter, H., Krob, E. J., Brooks, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+surface+properties+other+than+charge+involved+in+cell+separation+by+partition+in+polymer,+aqueous+2-phase+systems.">Membrane surface properties other than charge involved in cell separation by partition in polymer, aqueous 2-phase systems.</a> Biochemistry. 15 (14), 2959-2964 (1976).</li><li>Muzykantov, V. R., Murciano, J. C., Taylor, R. P., Atochina, E. N., Herraez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+complement-mediated+elimination+of+red+blood+cells+modified+with+biotin+and+streptavidin.">Regulation of the complement-mediated elimination of red blood cells modified with biotin and streptavidin.</a> Anal Biochem. 241 (1), 109-119 (1996).</li></ol>

관심 없음 충돌이 선언 없습니다.

유니버설 기증 적혈구는 수혈 치료를위한 강화 혈액 가용성 및 안전에 큰 잠재력을 지니고 있습니다. 적혈구는 오랜 순환 고유 biocompatibility 14, 15로 인해 유망 약물 투여 차량을 간주됩니다. 본 논문에서 제시 실험이 수정 적혈구를 HPG의 체외 특성에를 평가합니다. 체외의 속성과 HPG 수정 적혈구의 생체 유통은 최근 8, 11의 그룹에 조사되었습니다. NaCl과 인산 ?...

<table border="1"><tbody><tr><td> Glycidol </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> Trimethylolpropane </td><td> Fluka </td><td></td><td> (캐나다, ON) </td></tr><tr><td> 칼륨 methylate </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> 무수 피리딘 </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> 4 Dimethylaminopyridine </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> Succinic 무수물 </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> 아세톤 </td><td> 피셔 과학 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> 무수 디메틸 포름 아미드 </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> N-Hydroxysuccinimide </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> N, N&#39;-Diisopropylcarbodiimide </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> MTS 카드 </td><td></td><td></td><td> 마이크로 입력하면 시스템 (MTS) 카드 (FL, USA) </td></tr><tr><td> Dextran 500 kDa </td><td> Pharmacia 정밀 화학 </td><td></td><td> (스웨덴) </td></tr><tr><td> PEG 8 kDa </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td> FITC 단클론 항 리 서스 D (RhD) </td><td> 지수 Biodiagnostics </td><td></td><td> (PA, USA) </td></tr><tr><td> PE 단클론 항 CD47 </td><td> BD Biosciences </td><td></td><td> (NJ, USA) </td></tr><tr><td> Drabkin의 시약 </td><td> 시그마 알드리치 </td><td></td><td> (캐나다, ON) </td></tr><tr><td></td><td></td><td></td><td> 표. RBC 막과 분석 중합체를 HPG의 접목에 사용되는 화학 물질, 시약. </td></tr></tbody></table>

antigens protected functional red blood cells by the membrane grafting of compact hyperbranched polyglycerols

적혈구 (RBC) 수혈은 thalassemia 전공 및 낫 세포 빈혈 1-3으로 급성과 만성 의료 문제의 숫자의 치료를 위해 중요합니다. RBC 표면 (~ 308 항원 4) 만성 수혈 요법에 환자 transfused 적혈구 4, 5에 약간의 항원의 양을 일치로 인해 alloantibodies을 개발에 대한 항원의 다양한의 존재로 인해. 이러한 폴리에틸렌 글리콜 (PEG)와 hyperbranched polyglycerol (HPG)와 같은 친수성 고분자로 이식하는 등 산소, 포도당 및 이온 세와 같은 작은 분자의 흐름에 영향을주지 않고 표면 항원과 항체의 상호 작용을 방지 RBC 막에 제외 층을 형성한다. 현재 어떤 방법 때문에 적혈구 표면 및 D에 대한 항원 큰 수 (기반 단백질과 탄수화물)의 존재에 의해 제시 발굴 과제의 일환으로 보편적 적혈구 기증자 세포의 생성에 사용할 수 없습니다이러한 방법 evelopment이 크게 수혈의 안전을 향상시키고, 극적 적혈구의 가용성 및 사용을 향상시킬 수 있습니다. 이 보고서에서 HPG 및 특성의 접목 막에 의해 항원 보호 기능 적혈구를 개발하는 데 사용되는 실험이 제공됩니다. HPGs은 매우 컴팩트 한 폴리머 6, 7 biocompatible되며, 지질 막 8, 9 및 마스크 적혈구 표면 항원 10, 11을 둘러싸는 세포 glycocalyx 내에 위치 될 것으로 예상된다.

리 서스 D 항원과 CD47 RBC 표면 단백질의 위장복은 형광 표시 단클론 항체를 사용하여 유동 세포 계측법에 의해 정량화했고, 대표 결과는 그림 1에 있습니다. HPG - 이식 적혈구의 경우 (회색), 신호의 강도는 표면 단백질의 마스킹을 나타내는 세포 표면에 항체에 바인딩의 감소를 나타내는 컨트롤 적혈구 (적색 및 녹색)에 비해 (왼쪽으로 이동 피크) 감소 . <p class="jove_content" fo:keep-togeth...

Watch this Scientific Journal Video about Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols at JoVE.com

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

hyperbranched polyglycerol (HPG)와 적혈구의 세포막 수정 (적혈구)가 제공됩니다. 수정 적혈구는 수성 2 상 분할, 삼투 취약성과 보완 매개 용해에 의해 특징되었습니다. 표면 단백질과 항원의 위장은 유동 세포 계측법 및 마이크로 타이핑 시스템 (MTS) 혈액 phenotyping 카드를 사용 평가되었습니다.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

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 ...

antigens-protected-functional-red-blood-cells-membrane-grafting

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Immunology and Infection

18.0K 조회수.  University of British Columbia. hyperbranched polyglycerol (HPG)와 적혈구의 세포막 수정 (적혈구)가 제공됩니다. 수정 적혈구는 수성 2 상 분할, 삼투 취약성과 보완 매개 용해에 의해 특징되었습니다. 표면 단백질과 항원의 위장은 유동 세포 계측법 및 마이크로 타이핑 시스템 (MTS) 혈액 phenotyping 카드를 사용 평가되었습니다.

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

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

보완 고갈에 의해 특정 세포 집단의 고갈

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

연구

면역학 및 감염병학

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.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

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

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

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

확장, 정화, 그리고 사람 말초혈 NK 세포 기능 평가

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

Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric&#160;detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

Here we describe two assays for measuring complement activation induced by antibodies against red blood cells. The major advantage over the current assays is their quantitative and easy-to-interpret nature.

적혈구에 대한 항체에 의한 보체 활성의 정량 검출하는 방법

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

골수 세포 역학의 실시간 영상<em&gt; APC<sup&gt; 최소 / +</sup</em&gt; 소장 종양 디스크 공 초점 현미경을 회전시켜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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.

요오드 산 쉬프 염색 말초 혈액 단핵 세포에서 글리코겐을 감지

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

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

항원 중심의 성 대장염 모델의 개발은 항원이 T 세포에 세포를 제시하여 항원의 프리젠 테이션을 공부하기

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between the T-cell receptor and tumor antigens, presented by human leukocyte antigens, governs T-cell functionality; these methods include protein production, X-ray crystallography, biophysics, and functional T-cell experiments.

X 선 결정학, 생물 물리학 및 기능 분석 실험을 사용하면 메커니즘 집행 T 세포 암 항원의 수용체 인식을 확인하는 방법

Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell ...

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.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

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

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.