저자는 국방부를 통해 Congressionally 감독 의학 연구 프로그램 (# W81XWH-10-1-0445), 보건 국립 연구소 (NIH R21 HL110056)와 미국 심장 협회 (# 11SDG4890030)를 자금을 인정합니다.

1. 버퍼 및 시험 에이전트의 작성 및 살균 <ol><li> 150 MM NaCl 버퍼 : nanopure 물 500 ML에 4.383 g NaCl의 결정을 디졸브. </li><li> 산도 버퍼 : 일 염기의 및 이염 기성의 인산 나트륨의 적절한 양을 혼합하여 pH를 5.6, 6.2, 6.8 및 7.4에서 인산염 버퍼를 준비합니다. 샘플 등 구연산 버퍼로 다음 낮은 산도 값 (즉, 산도가 &lt;5.6) 더 적절한 버퍼에서 테스트 할 경우 사용되어야합니다. 버퍼 요리법은 쉽게 이용하실 수 있으며, 예를 들어 참조는 여기에서 제공되었습니다. 12 </li><li> 병 꼭대기에 진공 여과 장치를 다시 확인 버퍼 산도의를 통해 위에서 언급 한 모든 버퍼를 검사. </li><li> 20% 트리톤 X-100 (긍정적 제어) : 믹스 20 ML 순수 튼 nanopure 물 80 ML의 X-100. 힘차게 소용돌이과 분해하는 sonicate. 사용하기 전에 하룻밤 실온에서 둡니다. </li></ol> 2. 적혈구의 작성 <ol><li> 혈액 FR의 25 ML을 확보응고를 방지하기 K 2 - EDTA (에틸렌 다이아 민 테트라 초산) - 코팅 Vacutainer 튜브에 직접 그려 톰 익명의 인간 공여체. </li></ol> 참고 : 모든 절차는 해당 기관 검토위원회 (IRB) 및 venipuncture, 혈액 수집하여 사전 승인을 받아야합니다는 기증자에게 위험을 최소화하기 위해 훈련 phlebotomist가 수행해야합니다. 표준 정맥 절개 절차는 다른 출판되었습니다. 13 <ol start="2"><li> 5 분, 그리고 혈소판의 마크 수준 (빨강, 낮은 층) 및 플라즈마 (노란색, 상부 층) 관에 500 XG에서 혈액을 원심 분리기. </li><li> micropipettor을 통해 부드럽게 플라즈마를 대기음, 표백제로 추가 biohazardous 폐기물로 폐기하십시오. </li><li> 150 MM NaCl 용액으로 표시된 선 (플라즈마의 원래 수준)에 혈소판 관을 입력합니다. 모자와 부드럽게 혼합 할 수있는 몇 번 반전. 5 분 500 XG에 원심 분리기. </li><li> 다시 혈액 세포를 씻어 단계 2.3-2.4를 반복합니다. 그런 다음 supernat를 대기음개미와 산도 7.4에서 PBS로 교체하십시오. 혼합하는 반전. </li><li> 테스트 할 각 산도에 해당하는, 균일하게 사 튜브에 혈액을 분할. 테스트 할 각 산도 (5.6, 6.2, 6.8, 7.4)에 따라 튜브를 붙입니다. </li><li> 5 분 500 XG에서 혈액 튜브를 원심 분리기. 튜브의 레벨을 표시 한 후 표면에 뜨는 대기음. </li><li> 적절한 산도 (로 2.6에 표시된)의 버퍼로 표시 줄에 각 관을 입력합니다. </li><li> 라벨 네 50 ML 원뿔 튜브 (테스트에 산도를 하나씩), 각 원추형 튜브에 적절한 산도의 PBS의 pipet 49 ML. </li><li> 1시 50분 희석에 해당하는 관으로​​ 적혈구 (동일 PH) 1 ML을 추가합니다. 시각 흐린해야하며 방해받지 왼쪽하면 해결됩니다 희석 혈액을 검사한다. 더 펠렛 형태 경우, 세포는 lysed 있습니다. </li></ol> 3. 용해 분석 96 자 판 설치 및 정량화 <ol><li> 테스트 할 20x 원하는 최종 농도에서 모든 실험 약물 투여 요원 재고 솔루션을 준비 (검정약물 전달 에이전트의 10 μl + 최종 테스트 혼합물로 원래의 약물 전달 에이전트 20분의 1 희석으로 이어지는 190 μl 희석 적혈구를)됩니다. 20, 100, 및 800 μg / ML의 주식은 각각 1, 5, 40 μg / ML의 최종 테스트 농도의 결과로, 제안합니다. </li><li> V-하단에 각각의 재고 솔루션 96 - 웰 플레이트의 Pipet 10 μl. 최적의 결과를 얻으려면, 세중의 각 샘플을로드하거나 겹. </li></ol> 참고 : 쉽게, 그것은 별도의 96 - 웰 플레이트가 판 당 N에서 = 3-4로드 (각 농도에서) 각 샘플과 함께 테스트 할 각 산도를 준비해야하는 것이 좋습니다. <ol start="3"><li> 긍정적 인 제어 우물를 들어, 20 % 트리톤 X-100의 10 μl를 추가합니다. </li><li> 부정적인 제어 우물를 들어, 인산염 버퍼 10 μl를 추가합니다. 테스트 할 같은 산도에서 버퍼를 사용합니다. </li><li> 희석 적혈구의 Pipet 190 μl 각 우물 (2.10 참조), 남아 있는지 셀 재고 솔루션을전송 중에 균일. 힌트 :이 작업을 단순화하는 데 사용하는 멀티 채널 피펫. </li><li> 1 시간 (: 궤도 셰이커 또는 로커를 사용하여 선택 사항)을 37 ° C에서 접시를 품다. </li><li> 500 펠렛 그대로 적혈구에 XG에서 5 분에 대한 번호판을 원심 분리기. 참고 : 원심 분리기에서 판을 제거하고 다음 단계로 수송 할 때주의 처리하고 세포 펠렛을 방해하지 확신 할 수. </li><li> 멀티 채널 pipet 사용하여 깨끗하고 평평한 바닥 96 - 웰 플레이트에 각 우물에서 표면에 뜨는 100 μl를 전송합니다. 참고 : 셀 펠렛가 실수로 어떤 샘플 (들)에 대한 방해를하는 경우, 하나는 표면에 뜨는 이전이 진행 다음 판을 다시 원심 분리기 수 있습니다. </li><li> 플레이트 리더로 ​​supernatants의 흡광도를 측정합니다. 파장의 범위가 (- 541 nm의 400)로 사용할 수 있습니다. </li></ol> 참고 : 다른 판 독자들이 다른 채도 포인트와 민감한있을 수 있습니다, 그래서 나를 위해 파장의 선택asurements는 세제 치료에 의해 유도로 실험 샘플의 용혈 데이터를 안정적으로 최대 용혈에 대한 표준화 할 수 있는지 여부에 따라 달라집니다. 이 긍정적 인 컨트롤 샘플의 흡광도 값의 정확한 측정이 필요합니다. <ol start="10"><li> Microsoft Excel 또는 이와 유사한 데이터 분석 소프트웨어를 사용, 각 산도 (단계 3.4)에 대한 설정 대조군 샘플에서 배경 흡광도 판독의 평균을 찾습니다. 그 산도에서 측정 된 모든 다른 샘플에서이 배경 흡광도 값을 뺍니다. </li><li> 배경 제거 후, ​​긍정적 인 컨트롤의 평균 흡광도 세제 - 처리 샘플을 (단계 3.3)을 찾습니다. 그런 다음 100 % 용혈을 대표해야하는이 평균 흡광도 값으로 모든 실험 데이터 포인트를 정상화. 마지막으로, 세제 컨트롤도 상대적으로 각 개별에서 발생 %의 용혈을 계산하는 100 % 각각 잘 값을 곱합니다. </li></ol>

<ol>
	<li>Alberts, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biology of the Cell. , (2002).</li><li>Boasson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Bacteriolysis+by+Lysozyme.">On the Bacteriolysis by Lysozyme.</a> The Journal of Immunology. 34, 281-293 (1938).</li><li>Convertine, A. J., Benoit, D. S., Duvall, C. L., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+endosomolytic+diblock+copolymer+for+siRNA+delivery.">Development of a novel endosomolytic diblock copolymer for siRNA delivery.</a> J. Control. Release. 133, 221-229 (2009).</li><li>Duvall, C. L., Convertine, A. J., Benoit, D. S., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Delivery+of+a+Proapoptotic+Peptide+via+Conjugation+to+a+RAFT+Synthesized+Endosomolytic.">Intracellular Delivery of a Proapoptotic Peptide via Conjugation to a RAFT Synthesized Endosomolytic.</a> 7, 468-476 (2010).</li><li>Varkouhi, A. K., Scholte, M., Storm, G., Haisma, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endosomal+escape+pathways+for+delivery+of+biologicals.">Endosomal escape pathways for delivery of biologicals.</a> Journal of Controlled Release. 151, 220-228 (2011).</li><li>Plank, C., Oberhauser, B., Mechtler, K., Koch, C., Wagner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+endosome-disruptive+peptides+on+gene+transfer+using+synthetic+virus-like+gene+transfer+systems.">The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems.</a> Journal of Biological Chemistry. 269, 12918-12924 (1994).</li><li>Ratner, A. J., 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=Epithelial+Cells+Are+Sensitive+Detectors+of+Bacterial+Pore-forming+Toxins.">Epithelial Cells Are Sensitive Detectors of Bacterial Pore-forming Toxins.</a> Journal of Biological Chemistry. 281, 12994-12998 (2006).</li><li>Saar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-penetrating+peptides:+A+comparative+membrane+toxicity+study.">Cell-penetrating peptides: A comparative membrane toxicity study.</a> Analytical Biochemistry. 345, 55-65 (2005).</li><li>Kichler, A., Leborgne, C., Coeytaux, E., Danos, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-mediated+gene+delivery:+a+mechanistic+study.">Polyethylenimine-mediated gene delivery: a mechanistic study.</a> The Journal of Gene Medicine. 3, 135-144 (2001).</li><li>Behr, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Proton+Sponge:+a+Trick+to+Enter+Cells+the+Viruses+Did+Not+Exploit.">The Proton Sponge: a Trick to Enter Cells the Viruses Did Not Exploit.</a> CHIMIA International Journal for Chemistry. 51, 34-36 (1997).</li><li>Dawson, R. M. C., Elliot, D. C., Elliot, W. H., Jones, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Data for Biochemical Research. , (1986).</li><li>Ernst, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Applied Phlebotomy. , (2005).</li><li>Bulmus, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+pH-responsive+and+glutathione-reactive,+endosomal+membrane-disruptive+polymeric+carrier+for+intracellular+delivery+of+biomolecular+drugs.">A new pH-responsive and glutathione-reactive, endosomal membrane-disruptive polymeric carrier for intracellular delivery of biomolecular drugs.</a> Journal of controlled release : official journal of the Controlled Release Society. 93, 105-120 (2003).</li><li>Lackey, C. 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=Hemolytic+Activity+of+pH-Responsive+Polymer-Streptavidin+Bioconjugates.">Hemolytic Activity of pH-Responsive Polymer-Streptavidin Bioconjugates.</a> Bioconjugate Chemistry. 10, 401-405 (1999).</li><li>Murthy, N., Campbell, J., Fausto, N., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+pH-responsive+polymers+for+the+intracellular+delivery+of+biomolecular+drugs.">Bioinspired pH-responsive polymers for the intracellular delivery of biomolecular drugs.</a> Bioconjugate chemistry. 14, 412-419 (2003).</li><li>Murthy, N., Robichaud, J. R., Tirrell, D. A., Stayton, P. S., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+and+synthesis+of+polymers+for+eukaryotic+membrane+disruption.">The design and synthesis of polymers for eukaryotic membrane disruption.</a> Journal of controlled release : official journal of the Controlled Release Society. 61, 137-143 (1999).</li><li>Yu, H., 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=Overcoming+endosomal+barrier+by+amphotericin+B-loaded+dual+pH-responsive+PDMA-b-PDPA+micelleplexes+for+siRNA+delivery.">Overcoming endosomal barrier by amphotericin B-loaded dual pH-responsive PDMA-b-PDPA micelleplexes for siRNA delivery.</a> ACS nano. 5, 9246-9255 (2011).</li><li>Nelson, C. E., 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=Sustained+local+delivery+of+siRNA+from+an+injectable+scaffold.">Sustained local delivery of siRNA from an injectable scaffold.</a> Biomaterials. 33, 1154-1161 (2012).</li><li>Miozzari, G. F., Niederberger, P., H&#252;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+microorganisms+by+Triton+X-100.">Permeabilization of microorganisms by Triton X-100.</a> Analytical Biochemistry. 90, 220-233 (1978).</li><li>Chen, H., Zhang, H., McCallum, C. M., Szoka, F. C., Guo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unsaturated+Cationic+Ortho+Esters+for+Endosome+Permeation+in+Gene+Delivery.">Unsaturated Cationic Ortho Esters for Endosome Permeation in Gene Delivery.</a> Journal of Medicinal Chemistry. 50, 4269-4278 (2007).</li><li>Roth, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Measurements+and+Rational+Materials+Design+for+Intracellular+Delivery+of+Oligonucleotides.">Quantitative Measurements and Rational Materials Design for Intracellular Delivery of Oligonucleotides.</a> Biotechnology Progress. 24, 23-28 (2008).</li><li>Blumenthal, R., Seth, P., Willingham, M. C., Pastan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-dependent+lysis+of+liposomes+by+adenovirus.">pH-dependent lysis of liposomes by adenovirus.</a> Biochemistry. 25, 2231-2237 (1986).</li><li>Moore, N. M., Sheppard, C. L., Barbour, T. R., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+endosomal+escape+peptides+on+in+vitro+gene+delivery+of+polyethylene+glycol-based+vehicles.">The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles.</a> The Journal of Gene Medicine. 10, 1134-1149 (2008).</li><li>Panyam, J., Zhou, W. Z., Prabha, S., Sahoo, S. K., Labhasetwar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+endo-lysosomal+escape+of+poly(DL-lactide-co-glycolide)+nanoparticles:+implications+for+drug+and+gene+delivery.">Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery.</a> The FASEB Journal. 16, 1217-1226 (2002).</li></ol>

IRB 승인 : 인간의 과목을 포함하는 절차는 밴더빌트 대학 기관 심사위원회 (IRB, 프로토콜 # 111251)에 의해 승인되었습니다. 관심 없음 충돌이 선언 없습니다.

endosomolytic 기능을 위해 설계 산도 - 응답 고분자 또는 다른 요원이 신속하게 될 효과적으로 endosome에서 발생 산도 값의 붉은 혈액 세포의 용해 (그림 1을 기준으로 검사를 할 수 있습니다, 산도 6.8 - 초기 endosome, 산도 6.2 - 늦은 endosome, pH를 5.6 - lysosome). 14-17 산도에 따라 달라 용혈은 biomacromolecular의 치료학 (예 : 펩티드, siRNA, ODNs, 단백질)의 endosomal 버전을 중재 할 사업자의...

세포 내부에 많은 잠재적 영향력이 치료 대상이 있지만, 대리인의 세포 내 전달에 중요한 도전을 안겨주고 있습니다. 자주, 약물, 특히 biologics는 exocytosis를 통해 세포에 의해 internalized와 소포로 인신 매매 중 엔도-lysosomal 경로를 통해 내용의 저하로 연결, 또는 세포의 뒤쪽 shuttled 아르 수 있습니다. 후자의 과정에서 1 내부 pH는 소포의 효소의 활동을위한 최적의 산도입니다 약 5-6로 산성화되는 등 라이소자임 등이 칸에서 기능 2. 최근 자료 수는 특별히이 산성화가 자신의화물 cytosolic 전달을 촉진하기 endosomes 활용하여 설계되었습니다. 이 방법의 한 예는 그의 핵심 physiologic pH를 (예 : 7.4)에서 zwitterionic 및 충전 중립입니다 합성, 고분자 마이셀의 나노 입자를 사용합니다. 그러나, 산도 6.0에서- 6.5, 고분자는 protonated이되고 마이셀 코어를 destabilizes 그물 긍정적 인 요금을 습득하고, 노출 된 폴리머 세그먼트과 상호 작용할 수 있으며 endosomal 멤브레인을 방해. 이 활동은 그들의 cytosolic 목표에 액세스 할 수 있습니다 펩타이드 및 핵산 기반 치료학의 endosomal 탈출을 홍보하기 위해 표시되었습니다. 막 장벽을 방해 endosomal 탈출을 중재하기 위해 개발 방법의 3,4 다른 예는 또는 &#39;fusogenic&#39;펩티드를 포함 인지질 이중층에 막 융합 또는 과도 기공 형성을 중재 할 수 있습니다 단백질이. 같은 폴리 (propylacrylic 산)와 같은 음이온 알킬 아크릴 산의 5 Homopolymers 또 다른 잘 공부 접근 방법이며, 이러한 고분자에 펜던트 카르 복실 산의 protonation 상태를 전환 해주는 엔도-lysosomal 산도 범위에서 소수성으로, 막 중단 상태입니다. 6,7 endosomolytic 행동을 검사에 대해 하나의 유용한 모델 시스템은 전자입니다X 생체의 산도에 의존 용혈 분석 8.이 모델 시스템에서, 적혈구 막에는 엔도-lysosomal 소포를 동봉 지질 이중층 막에 대한 대리 역할을합니다. 이 generalizable 모델이 세포 관통 펩티드 및 기타 고분자 유전자 전달 시스템의 endosomolytic 행동을 평가하는 다른 사용되었습니다. 8-11이 실험에서 인간의 적혈구와 테스트 자료는 모방 정의 PHS에서 버퍼에 공동 incubated 아르 세포 (7.4), 초 (6.8) endosomal, 그리고 엔도-lysosomal 하순 (&lt;6.8) 환경. 부화 기간 동안 출시 된 헤모글로빈의 양이 세제로 lysed 긍정적 인 제어 샘플에 출시 헤모글로빈의 양에 표준화되어 적혈구 용해의 측정으로 정량화되어 있습니다. 잠재적 endosomolytic 시험 자료의 작은 도서관을 심사에서, 하나는 산도 7.4에서 더 용혈을 생산하지 그 샘플을 추론하지만, 상당히 고농도의 헛기침 수산도 &lt;6.5에서 olysis은 cytosolic 약물 전달을위한 가장 효과적이고 cytocompatible 후보 될 것입니다. 이 기준에 맞는 자료를 무차별 엔도-lysosomal 구획에 국제화에 따라 지역의 pH의 감소에 노출되는 때까지 지질 이중층의 세포막 (세포 독성을 일으킬 수 IE)를 파괴 불활성이 아닌 상태로 유지 될 것으로 예상됩니다. 이 프로토콜에서는 적혈구는 인간의 기증자로부터 분리되며, 산도 5.6, 6.2, 6.8, 또는 실험 endosomolytic 약물 전달 에이전트와 7.4에서 공동 incubated. 그대로 적혈구는 pelleted 있으며, supernatants은 (lysed 적혈구에서 출시 헤모글로빈을 포함) 플레이트 리더 (그림 1)를 통해 헤모글로빈의 특성 흡광도를 분석하고 있습니다.

<table border="1"><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> BD Vacutainer - K2EDTA Vacutainer 튜브 </td><td> 피셔 과학 </td><td> 22-253-145 </td><td> 혈액 수집을위한 </td></tr><tr><td> BD Vacutainer 혈액 수집 바늘, 20.5 게이지 </td><td> 피셔 과학 </td><td> 02-665-31 </td><td> 혈액 수집을위한 </td></tr><tr><td> BD Vacutainer 튜브 홀더 / 니들 어댑터 </td><td> 피셔 과학 </td><td> 22-289-953 </td><td> 혈액 수집을위한 </td></tr><tr><td> BD 브랜드 이소 프로필 알코올 스왑 </td><td> 피셔 과학 </td><td> 13-680-63 </td><td> 혈액 수집을위한 </td></tr><tr><td> BD Vacutainer 라텍스 무료 지혈대 </td><td> 피셔 과학 </td><td> 02-657-6 </td><td> 혈액 수집을위한 </TD&gt; </tr><tr><td> 염산 (conc.) </td><td> 피셔 과학 </td><td> A144-500 </td><td> D-PBS의 산도 조정하십시오. </td></tr><tr><td> 트리톤 X-100 </td><td> 시그마 - 알드리치 </td><td> T8787 </td><td> 긍정적 인 제어 </td></tr><tr><td> Dulbecco의 PBS </td><td> Invitrogen </td><td> 14,190 </td><td></td></tr><tr><td> SFCA 막과 Nalgene MF75 무균 일회용 병 - 상위 필터 단위 </td><td> 피셔 과학 </td><td> 09-740 - 44A </td><td></td></tr><tr><td> BD 96 - 웰 플레이트, 평면 바닥, 조직 문화 처리 폴리스티렌 </td><td> 피셔 과학 </td><td> 08-772 - 2C </td><td> 분석의 끝에서 판 읽기하십시오. </td></tr><tr><td> BD 96 - 웰 플레이트, 둥근 바닥, 조직 문화 처리 폴리스티렌 </td><td> 피셔 과학 </td><td> 08-772-17 </td><td> 실험 대리인과 붉은 혈액 세포의 배양하십시오. </td></tr></표&gt;

ex vivo red blood cell hemolysis assay for the evaluation of ph-responsive endosomolytic agents for cytosolic delivery of biomacromolecular drugs

엔도-lysosomal 소포를 구성 인지질의 bilayers은 세포 내 표적에 대한 생물학적 약물의 전달에 장벽을 야기 할 수 있습니다. 이 장벽을 극복하기 위해, 합성 약물 캐리어의 수는 적극적으로 endosomal 멤브레인을 방해하고 세포질로화물을 제공하도록 설계되었습니다. 여기, 우리는 세포 내 약물 전달 시스템의 cytocompatibility 및 endosomolytic 활동에 대한 신속하고 높은 처리량 화면으로 사용할 수있는 용혈 분석을 설명합니다. 용혈 분석에서 인간의 적혈구와 테스트 자료는 세포, 초기 endosomal, 늦은 엔도-lysosomal 환경을 모방 정의 PHS의 버퍼의 Co-incubated 수 있습니다. 펠렛 그대로 적혈구에 원심 분리 단계 후, 매체에 발표 헤모글로빈의 양이 spectrophotometrically (가장 동적 범위 405 nm의) 측정됩니다. %의 적혈구 장애는 이후 긍정적 인 제어 samp에 상대적으로 정량화된다레 세제로 lysed. 이 모델 시스템에서 적혈구 막에는 엔도-lysosomal 소포를 동봉 지질 이중층 막에 대한 대리 역할을합니다. 원하는 결과는 physiologic pH를 (7.4) 약 산도 5-6.8에서 엔도-lysosomal 산도 범위에서 강력한 용혈에서 무시할 용혈 수 있습니다.

<html> 일반적으로 이상적인 산도에 의존 용혈성 문제가 발생할 요원이 마약, 핵산, 또는 다른 bioactive 분자의 cytosolic 제공을위한 최고의 잠재력을 갖추고 있습니다. 이것은 산도 7.4에서 최소한의 용혈을 전시하고 그림 2에 묘사로 에이전트 # 1 예시하지만, endosomal 산도 범위 (&lt;6.5)에서 용혈성 행동의 날카로운 증가하고 있습니다. 이러한 대리인이 hemocompatible되지 않을 수 있습니다 잠재...

Watch this Scientific Journal Video about Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs at JoVE.com

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

용혈 분석은 세포 내화물 배달 약물 전달 시스템 &#39;cytocompatibility과 endosomolytic 활동의 빠른, 높은 처리량 화면으로 사용할 수 있습니다. 검정은 환경 산도의 함수로 적혈구 멤브레인의 중단을 측정합니다.

Biomacromolecular 일반 의약품의 Cytosolic 전송 산도 - 응답 Endosomolytic 에이전트의 평가에 대한 예 VIVO 레드 블러드 세포 용혈 분석

Phospholipid bilayers that constitute endo-lysosomal vesicles can pose a barrier to delivery of biologic drugs to intracellular targets. To overcome this ...

ex-vivo-red-blood-cell-hemolysis-assay-for-evaluation-ph-responsive

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JoVE Journal

Immunology and Infection

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

34.0K 조회수.  Vanderbilt University. 용혈 분석은 세포 내화물 배달 약물 전달 시스템 'cytocompatibility과 endosomolytic 활동의 빠른, 높은 처리량 화면으로 사용할 수 있습니다. 검정은 환경 산도의 함수로 적혈구 멤브레인의 중단을 측정합니다.

비디오: Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

연구

면역학 및 감염병학

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokinetic 트랙 운동성 분석에 의한 세포 마이그레이션의 수량 평가

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

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.

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 ...

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 ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

높은 처리량 정량 실시간 RT-PCR 분석 유형 I과 III 인터페론 하위 유형의 발현 양상을 결정하는

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

단핵구 단층 분석의 사용은 Fcγ 수용체 - 매개 식균 작용을 평가하기

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

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 ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new method with a powerful and simple strategy for the establishment of B-LCLs, the red blood cell lysis method. This method simplified the PBMC separation procedure with red blood cell removal, and used as little as 0.5 mL of whole blood for establishing EBV-immortalized cell lines, which can proliferate to large cell numbers in a relatively short amount time with a 100% success rate. The method is simple, reliable, time saving, and applicable to treating a large number of the clinical samples.

A simple red blood cell lysis method for establishing B-LCLs was developed with high immortalization efficiency, requiring a small amount of blood and saving time from initiation to cryopreservation.

B 임파 세포 라인의 설립에 대한 간단한 적혈구 용해 방법

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Vivo에서 예방 접종 모델을 사용 하는 항 원 특정 T-셀 Cytolytic 함수의 검색에 대 한 분석 결과

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Deformability 및 Ektacytometry에 의해 혈액이 질 레드 셀 측정

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...