This work was supported by the Seed Grant Award from California Northstate University College of Pharmacy (to AMN). The authors would like to thank California Northstate University College of Pharmacy for covering the publication cost of this article, and George Talbott for his help in editing this manuscript.

카코 2 셀 1. 유지 보수 <ol><li> 일반 조직 배양 접시를 사용하여 <ol><li> (FBS를 37 ℃의 수조에서 바이알을 배치하여 냉동 바이알로부터의 카코 -2 세포를 해동하고, 즉시 예열 된 성장 배지 (15 % 소 태아 혈청 10 ㎖를 함유 10cm 조직 배양 접시에 추가 ) 둘 베코의 수정 이글 중간 (DMEM))에서. <ol><li> 카코 -2 세포 50~70% 합류점에 도달 한 경우, 이들을 1 분할 : 그들은 분리 될 때까지 37 ℃에서 0.05 % 트립신 / 0.53 mM의 에틸렌 디아민 테트라 아세트산 (EDTA) 3 ㎖와 함께 세포를 배양하여 6 (15 분) . 세포 응집을 방지 부드러운 피펫 팅하여 세포를 여러 번 혼합합니다. 예열 성장 배지 10 ㎖를 함유 10cm 조직 배양 접시에 세포를 트립신 0.5ml를 추가. </li></ol></li><li> 부드럽게 좌우 방향으로 이어 전후 방향으로 요리를 여러 번 흔들어. 요리를 소용돌이 피으로는 불평등 한 세포 분산 될 수 있습니다. </li><li> 5 % CO 2와 함께 제공되는 37 ° C의 배양기에서 평평한 표면에 요리를 놓습니다. 경사면은 불평등 세포 분산 될 수 있습니다. </li><li> 배양을 시작한 후 성장 미디어에게 일을 변경합니다. </li><li> 세포를 모니터링하고 그들이 합류에 도달 하루를 기록한다. 셀들이 분할 일로부터 합류에 도달하는 것은 약 1 주일 정도 걸릴 것이다. </li><li> 세포가 합류에 도달하기 전에 일주일에 두 번 성장 미디어를 변경합니다. 세포가 합류에 도달하면, 매일 성장 미디어를 변경합니다. 세포가 7 일 후 합류 한 후 매일 성장 미디어를 변경합니다. 주 :이 13 일 후 합류 (그림 1) 때 세포 실험에 대한 준비가되어 있습니다. 세포는 결국 지질 단백질 생산에 덜 효과적이 될 것이기 때문에, 8 포스트 합류 13-17 - 일 사이에 세포를 사용합니다. 실험을 수행하는 방법에 대한 3 단계로 이동합니다.</li></ol></li><li> 투과 막 시스템을 사용하여 <ol><li> 단계 1.1.1.1에 기재된 투과성 막 인서트 카코 -2 세포를 시드. 위. 자신의 휴대 성장 (회복 기간) 일반적으로 느린 때문에 냉동 유리 병에서 세포를 사용하지 마십시오. 간단히, 미리 예열 성장 배지 10 ㎖를 포함하는 혀끝 챔버 (상단 구획)에 트립신 세포의 0.5 ml를 추가합니다. 또한, 기저 챔버 (낮은 구획)에 미리 예열 성장 배지 10 ㎖를 추가합니다. 참고 : 가장 좋은 방법은, 기저 챔버 먼저 다음 혀끝 챔버로 성장 매체를 추가 할 수 있습니다. 이전 용지를 제거 할 때, 혀끝의 미디어하기 전에 기저 용지를 제거합니다. 이 막 파열 될 수 있으므로 폴리 카보네이트 막 파고하지 마십시오. </li><li> 인하여 폴리 카보네이트 막을 통해 열악한 셀 가시성 유사한 밀도 정규 조직 배양 접시에 카코 -2 세포를 시드 및 셀 합류 판단이 접시를 사용한다. </li><li> 의에 따라TEPS 1.1.2-1.1.6 위. </li></ol></li></ol> 2. 유전자 과발현 <ol><li> 일반 형질 전환 방법을 사용하여 <ol><li> 상술 1.1.1.1.-1.1.4에 기술 된 바와 같이 조직 배양 접시에 카코 -2 세포를 시드. 주 : 세포가 40-50 %의 컨 플루 언트 될 때, 형질 감염이 될 준비가된다. </li><li> microcentrifuge 관에서 감소 된 혈청 미디어의 472 μL에 형질 전환 시약의 67 μl를 추가합니다. 튜브 벽 희석 형질 전환 시약의 접촉을 피하십시오. </li><li> pLL3.7 23 μg의이 형질 전환 시약 / 감소 혈청 미디어 혼합물에 녹색 형광 단백질 (eGFP는) 9 향상된 추가합니다. </li><li> RT에서 20 분 동안 혼합물을 인큐베이션. </li><li> DMEM에 10 % FBS 8 mL로 카코 2 세포의 성장 매체를 교체합니다. </li><li> 드롭 현명한 접시에 DNA / 형질 전환 시약 / 감소 혈청 미디어 혼합물을 추가합니다. </li><li> 조심스럽게 앞으로 바에서 요리를 여러 번 흔들어좌우 방향으로 이어 ckward 방향. </li><li> 37 ° C 배양기에서 접시를 놓습니다. </li><li> 배양을 시작한 후 성장 배지 10 ml의 하루 접시에 미디어를 교체합니다. </li><li> 유전자 발현을 모니터링합니다. <ol><li> 4 &#39;, 6-diamidino -2- 페닐 인돌 (DAPI) 얼룩없이 유전자 발현을 모니터한다. 형광 현미경을 사용하여, 녹색 세포의 비율을 결정한다. 비율은 형질 전환 효율을 나타낸다. </li><li> DAPI 염색과 유전자 발현을 모니터링합니다. <ol><li> 인산염 완충 식염수 (PBS)로 3 회 세포를 씻으십시오. </li><li> PBS (RT에서 10 분 동안 배양)에서 4 % 포름 알데히드를 10 ㎖를 첨가하여 세포를 고정. </li><li> PBS로 3 회 세포를 씻으십시오. </li><li> DAPI (500), 1 % BSA, 0.01 %의 디기 토닌 : 1을 함유하는 PBS의 8 mL를 실온에서 15 분 동안 어둠 속에서 세포를 배양한다. </li><li> PBS로 3 회 세포를 씻으십시오. </li><li> 유형광 현미경을 노래 블루 (그림 2 상단 패널) 된 것을 녹색 / 청색 상대적 세포의 수를 결정한다. 계산 된 비율은 형질 감염 효율을 나타낸다. </li></ol></li></ol></li></ol></li><li> 렌티 바이러스 과발현 시스템 (10)을 사용하여 <ol><li> 15cm 조직 배양 접시에 씨앗 인간 배아 신장 (HEK) 293T 세포 (DMEM에 10 % FBS 성장 매체로). 주 : 세포가 60-70 %의 컨 플루 언트 될 때, 형질 감염이 될 준비가된다. </li><li> microcentrifuge 관 (11)에 감소 된 혈청 매체의 1,133 μL에 형질 전환 시약 162 μl를 추가합니다. 튜브 벽 희석 형질 전환 시약의 접촉을 피하십시오. </li><li> 추가 24 pLL3.7 eGFP는 (또는 다른 구조물)의 μg의 15.6 레브 반응 요소 μg의 (RRE) REV 6.0 μg의 한 형질 감염 시약 / R로 포성 구내염 바이러스 당 단백질 8.4 μg의 (VSVG)educed 혈청 미디어 혼합물. </li><li> RT에서 20 분 동안 혼합물을 인큐베이션. </li><li> DMEM에 10 % FBS 16 mL로 성장 매체를 교체합니다. </li><li> 드롭 현명한 접시에 DNA / 형질 전환 시약 / 감소 혈청 미디어 혼합물을 추가합니다. </li><li> 부드럽게 좌우 방향으로 이어 전후 방향으로 요리를 여러 번 흔들어. 이 시점에서 분리 HEK293T 세포의 일부를 참조하는 정상이다. </li><li> 인큐베이터에서 접시를 놓습니다. </li><li> 배양 24 시간 후, 성장 배지 25 mL를 접시에 미디어를 교체합니다. </li><li> 다음 날, 렌티 바이러스 (제 컬렉션) 함유 증식 배지를 수집한다. </li><li> 단계를 반복 2.2.10 더 렌티 바이러스 (두 번째 컬렉션)를 수집합니다. </li><li> (5 분 동안 2,500 XG) 제 1 및 제 2 집합을 수영장, 원심 분리에 의해 세포 파편을 제거한다. </li><li> 일회용 병 톱 필터 (0.45 μm의 기공)과 컬렉션을 필터링하고,RT (JA-20 로터)에서 2 시간 동안 31,400 XG에서 컬렉션을 원심 분리하여 바이러스를 농축시킨다. 펠렛이있는 원심 분리 튜브의 하단을 표시한다. 상등액을 경사 분리하고, 약 15 분 동안 RT에서 반전 튜브를 떠난다. </li><li> 1X PBS 100 ㎕와 바이러스 - 함유 펠렛을 재현 탁. 거품을 피하십시오. </li><li> -80 ° C에서 집중 바이러스를 저장합니다. 동결 - 해동 사이클을 피하십시오. </li><li> 적정에 의해 카코 2 세포를 형질 도입에 필요한 바이러스의 최적의 양을 결정합니다. 비용을 절약하기 위해, 24 웰 플레이트 (물론 성장 미디어 / 0.3 ml)을 대신 적정 10cm 조직 배양 접시를 사용합니다. <ol><li> 농축 된 바이러스의 양을 증가시키는 추가의 (0, 1, 2, 5, 10, 25, 50 μL / 웰) 40~70% 합류 카코 -2 세포에 폴리 브렌 (최종 농도 = 5 μg의 / ㎖)으로 보충. 1.1.2에 설명 된 바와 같이 부드럽게 24- 웰 플레이트를 흔들어. </li><li> 자신의 유전자 발현 모니터 (단계 2.1.10를.) (그림 2 바닥4 패널). </li></ol></li><li> 형질 전환 유전자의 발현은 일반적으로 유지되어 있기 때문에, 미래의 실험 형질 세포를 유지하고 지속적으로 유전자 발현을 모니터한다 (단계를 2.1.10.). </li></ol></li></ol> 3. 지단백질 분비를 자극 <ol><li> 일반 조직 배양 접시를 사용하여 <ol><li> 올레산 50 ㎎, 레시틴 40 mg의 나트륨 타우로 콜산 48 mg을 혼합하여 100X 지질 혼합물을 제조한다. (8 요리에 대한 충분한) PBS로 900 μL에 볼륨을 가져와. 적극적으로 지질 혼합물을 소용돌이. </li><li> 성장 미디어의 89.1 ml의에 100X 지질 혼합물의 900 μl를 추가합니다. 혼합 및 지질 함유 미디어를 필터링 할 수 있습니다. 올레산, 레시틴, 및 나트륨 타우로 콜산의 최종 농도 (1X)는 2.0mm의 1.36 mm의 각각 1.0 mm의이다. </li><li> 세포에 지질 함유 미디어의 10 ML을 추가합니다. </li><li> (37)에서 4 시간 동안 지질 함유 매체를 사용하여 세포를 품어C 배양기를 °. </li><li> PBS로 3 회 세포를 씻으십시오. </li><li> 지질 단백질의 분비를 수집 세포 성장 배지 10 ㎖를 추가한다. </li><li> 37 ° C 배양기에서 2 시간 동안 품어. </li><li> 지질 단백질 함유 미디어를 수집합니다. </li></ol></li><li> 투과 막 시스템을 사용하여 <ol><li> 단계에 따라 3.1.1-3.1.2 지질 혼합물을 제조. </li><li> 혀끝 챔버 지질 함유 미디어 10 ㎖ 및 기저 챔버로 성장 매체의 10 ML을 추가합니다. </li><li> 37 ° C 배양기에서 4 시간 동안 품어. 기저 챔버에서 지질 단백질 함유 미디어를 수집합니다. </li></ol></li></ol> 4. 지단백 분리 (그림 3) <ol><li> 원심 분리 (5 분 2,000 XG)에 의해 지질 단백질 함유 매체에서 세포 파편을 제거합니다. </li><li> 50 ㎖ 튜브에 미디어를 가만히 따르다. </li><li> 미디어에 염화나트륨 5.95 g을 넣고. </li><li> SA의 경우mple이 TEM에 의해 분석되며, 지단백질의 무결성을 유지하기 위해 1, 프로테아제 억제제 칵테일 정제를 추가한다. </li><li> 물 23 ㎖의에 볼륨 (1.2 ㎍ / ㎖의 NaCl 농도 솔루션)를 준비한다. </li><li> 완전히 용질을 녹여. </li><li> 폴리 카보네이트 초 원심 분리기 튜브에 모든 용액을 경사 분리한다. </li><li> 부드럽게 0.5 ml의 물 (1.0 ㎍ / ㎖ 농도)와 1.2 ㎍ / ㎖ 밀도 솔루션을 오버레이. </li><li> 부피 중량이 아닌 튜브를 균형. </li><li> 조심스럽게 T865 회 전자에 튜브를로드합니다. </li><li> 4 ℃에서 24 시간 동안 429,460 XG에서 초 원심 분리기에서 샘플을 스핀. </li><li> 즉시 부드러운 피펫으로 최고 0.5 ml의 솔루션을 격리 할 것. 가능한 한 여전히 튜브를 유지합니다. </li></ol> 5. TEM 분석 <ol><li> (W / V) 및 6.0의 pH를 조절 2 % 포스 포 텅스텐 산을 5 ml를 준비한다. </li><li> (0.2 ㎛의 공극 크기) 주사기 필터와 2 % 인 텅스텐 산 필터. </li><li> 멸균 접시에 지질 단백질 샘플의 20 μl를 삭제합니다. </li><li> 다음 같은 접시에 샘플로 필터링 2 % 인 텅스텐 산의 20 μl를 삭제합니다. </li><li> 조심스럽게 무딘면이 샘플에 휴식과 EM 그리드를 놓습니다. </li><li> 1 분 동안 실온에서 품어. </li><li> 조심스럽게 그리드에서 샘플을 제거하기 위해 필터 종이에 EM 그리드의 측면을 누릅니다. </li><li> 조심스럽게 무딘면이 2 % 인 텅스텐 산에 휴식과 EM 그리드를 놓습니다. </li><li> 1 분 동안 실온에서 품어. </li><li> 조심스럽게 그리드에서 포스 산을 제거하기 위해 필터 종이에 EM 그리드의 측면을 누릅니다. </li><li> TEM을 사용하여, 지단백질 (도 4)의 이미지를 캡쳐. </li></ol>

<ol>
	<li>Drover, V. 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=CD36+deficiency+impairs+intestinal+lipid+secretion+and+clearance+of+chylomicrons+from+the+blood.">CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.</a> J Clin Invest. 115 (5), 1290-1297 (2005).</li><li>Nauli, A. M., 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=CD36+is+important+for+chylomicron+formation+and+secretion+and+may+mediate+cholesterol+uptake+in+the+proximal+intestine.">CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine.</a> Gastroenterology. 131 (4), 1197-1207 (2006).</li><li>Nauli, A. M., Zheng, S., Yang, Q., Li, R., Jandacek, R., Tso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+release+is+not+associated+with+chylomicron+formation.">Intestinal alkaline phosphatase release is not associated with chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 284 (4), G583-G587 (2003).</li><li>Lo, C. -. M., 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=Why+does+the+gut+choose+apolipoprotein+B48+but+not+B100+for+chylomicron+formation.">Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 294 (1), G344-G352 (2008).</li><li>Jandacek, R. J., Heubi, J. E., Tso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+noninvasive+method+for+the+measurement+of+intestinal+fat+absorption.">A novel, noninvasive method for the measurement of intestinal fat absorption.</a> Gastroenterology. 127 (1), 139-144 (2004).</li><li>Nauli, A. M., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+transport+as+a+potential+determinant+of+drug+bioavailability.">Intestinal transport as a potential determinant of drug bioavailability.</a> Curr Clin Pharmacol. 8 (3), 247-255 (2013).</li><li>Tso, P., Nauli, A., Lo, 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=Enterocyte+fatty+acid+uptake+and+intestinal+fatty+acid-binding+protein.">Enterocyte fatty acid uptake and intestinal fatty acid-binding protein.</a> Biochem Soc Trans. 32 (Pt 1), 75-78 (2004).</li><li>Nauli, A. M., Sun, Y., Whittimore, J. D., Atyia, S., Krishnaswamy, G., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chylomicrons+produced+by+Caco-2+cells+contained+ApoB-48+with+diameter+of+80-200+nm.">Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.</a> Physiol Rep. 2 (6), (2014).</li><li>Rubinson, D. 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=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33 (3), 401-406 (2003).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Pottekat, 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=Insulin+biosynthetic+interaction+network+component,+TMEM24,+facilitates+insulin+reserve+pool+release.">Insulin biosynthetic interaction network component, TMEM24, facilitates insulin reserve pool release.</a> Cell Rep. 4 (5), 921-930 (2013).</li><li>Van Greevenbroek, M. M., van Meer, G., Erkelens, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+saturated,+mono-,+and+polyunsaturated+fatty+acids+on+the+secretion+of+apo+B+containing+lipoproteins+by+Caco-2+cells.">Effects of saturated, mono-, and polyunsaturated fatty acids on the secretion of apo B containing lipoproteins by Caco-2 cells.</a> Atherosclerosis. 21 (1), 139-150 (1996).</li><li>Levy, E., Yotov, W., Seidman, E. G., Garofalo, C., Delvin, E., M&#233;nard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cells+and+human+fetal+colon:+a+comparative+analysis+of+their+lipid+transport.">Caco-2 cells and human fetal colon: a comparative analysis of their lipid transport.</a> Biochim Biophys Acta. 1439 (3), 353-362 (1999).</li><li>Luchoomun, J., Hussain, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+secretion+of+chylomicrons+by+differentiated+Caco-2+cells.+Nascent+triglycerides+and+preformed+phospholipids+are+preferentially+used+for+lipoprotein+assembly.">Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly.</a> J Biol Chem. 274 (28), 19565-19572 (1999).</li></ol>

The author has nothing to disclose.

본 논문에서는 사용될 수 개의 시스템 카코 -2 세포, 즉, 정규 조직 배양 접시 투과 막을 기재되어 유지한다. 투과성 막 시스템을 사용할 때의 이점은 정점의 분리 및 기저 외측 구획하고, 지질 혼합물을 배양하는 동시에 지단백질 분비를 수집하는 기능을 포함한다. 다만, 투과 막 인서트 비싸고, 그 폴리 카보네이트 막 좋은 셀 가시성을 허용하지 않는다. 이 시험 관내 모델의 장점 중 하나는...

4 -식이 지방과 지용성 약물과 비타민의 장내 흡수 연구는 림프 누공 모델 1을 사용하여 생체 내에서 수행 할 수있다. 그러나 관련된 수술 기법은 도전뿐만 아니라 비용이 많이 드는되지 않습니다. 생체 이용 될 수 분변 분석에 기반 방식이지만, 이들은 위장관 2,5- 의해 흡수 퍼센트를 결정 주로 사용된다. 이 문서에 설명 된 체외 모델은 비용 효과적이며, 관련 기술은 틀림없이 덜 도전이다. 유전자 변형 연구는 시간이 많이 걸리는 사람들이 체외 모델을 사용하여 수행 할 때 더 경제적이고 저렴합니다. 장 세포에 의해 흡수되는 지용성 물질 지단백질 -6,7-로 포장되어 있기 때문에, 리포 단백질을 생산하기 위해이 시험 관내 모델의 효과는 매우 중요하다. 두 가지 주요 intestinaL 지단백질은 카일로 마이크론과 매우 저밀도 지단백질 (VLDL)입니다. 지질은 위장 루멘에 풍부하게 존재하는 경우 직경이 80 nm 이상과 지질 단백질로 정의 카일로 마이크론은, 소장에 의해 엄격하게 생산된다. 그들이 가장 큰 지단백질이기 때문에, 카일로 마이크론은 생각할 가장 효율적인 지질 수송이다. 킬로 미크론 (8)을 제조 할 수있는 이러한 시험 관내 모델은,식이 지방 흡수에 의한 소화관 지용성 비타민의 흡수 및 지용성 약물 경구 생체 이용률을 연구하는데 사용될 수있다. 지단백질 분획에 지용성 분자, 비타민, 또는 약물의 존재는 소장 흡수하여 자신의 지표이다. 전술 한 바와 같이,이 모델은 경구 지용성 약물 생체 이용율 (6)을 향상 시키는데 사용될 수있다. 이 논문은 카코 2 세포 투과 막 또는 일반 조직 배양 접시, 어떻게 지질 마일에서 유지되어야한다 방법을 설명합니다지질 단백질 생산을 자극 xture는 렌티 바이러스 발현 시스템이 효율적인 과발현을 달성하기 위해 이용 될 수있는 방법을 준비해야하며, 절연 지단백질은 어떻게 분석되어야한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>16750-112</td>
			<td>Pre-warm the growth media in individual tissue culture dish before adding cells</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Fisher</td>
			<td>3600511</td>
			<td>We do not heat inactivate our serum</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-660</td>
			<td>Cells can be washed with PBS prior to trypsin treatment&#160;</td>
		</tr>
		<tr>
			<td>Permeable membrane system</td>
			<td>Fisher</td>
			<td>7200173</td>
			<td>10-cm dish, 3-mm pore size, polycarbonate membrane</td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Fisher</td>
			<td>08-772-E</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>15 cm dish</td>
			<td>Fisher</td>
			<td>08-772-24</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Fisher</td>
			<td>12565163</td>
			<td>Tissue culture plate</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Fisher</td>
			<td>PRE2691</td>
			<td>Can be substituted with other transfection reagent</td>
		</tr>
		<tr>
			<td>Reduced serum media</td>
			<td>Invitrogen</td>
			<td>11058021</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>pLL3.7 eGFP</td>
			<td>Addgene</td>
			<td>11795</td>
			<td><a href="https://www.addgene.org/11795/">https://www.addgene.org/11795/</a></td>
		</tr>
		<tr>
			<td>Bottle-top filter</td>
			<td>Fisher</td>
			<td>9761120</td>
			<td>0.45 mm pore</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Fisher</td>
			<td>NC9840454</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma</td>
			<td>01383-5G</td>
			<td>Prevent freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>Lecithin&#160;</td>
			<td>Fisher</td>
			<td>IC10214625</td>
			<td>Egg lecithin&#160;</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Fisher</td>
			<td>NC9620276</td>
			<td>Product discontinued; alternative catalog number: 50-121-7956</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablet (EDTA-free)</td>
			<td>Fisher</td>
			<td>5892791001</td>
			<td>Used mainly for samples that need TEM analysis</td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9696153</td>
			<td>Reusing it multiple times will collapse the tube</td>
		</tr>
		<tr>
			<td>Lid for ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9796914</td>
			<td>A tool is required to remove the tube/lid from rotor</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher</td>
			<td>50-949-261</td>
			<td>Disposable</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Fisher</td>
			<td>09-719C</td>
			<td>Pore size = 0.2 mm; nylon</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Fisher</td>
			<td>AC208310250</td>
			<td>For preparing 2% phosphotungstic acid, pH 6.0</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Fisher</td>
			<td>50-238-62</td>
			<td>Extra fine and strong tips</td>
		</tr>
		<tr>
			<td>Formvar/carbon grid</td>
			<td>Fisher</td>
			<td>50-260-34</td>
			<td>Formvar/carbon film square grid 400 Copper</td>
		</tr>
	</tbody>
</table>

using caco-2 cells to study lipid transport by the intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

1 표시 정상 13 일 이후 합류 카코 2 세포를 그림. 돔형 구조 및 세포 내 지질 액 적의 모습 분화 카코 -2 세포의 특성이다. 카코 2 세포를 파종시에 동일하게 분산되지 않을 때, 그들은 덩어리와 접시의 특정 지역에서 자라다 것이다; 어떤 세포없이 접시에 몇 가지 영역이 될 것입니다. 소용돌이 경사면에 접시를 배치하는 것은 피해야한다. 이 후 합류 카코 2 세포가 새로운 ?...

Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

소장에 의해 지질 전송을 연구하기 위해 카코 2 셀을 사용하여

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

using-caco-2-cells-to-study-lipid-transport-by-the-intestine

Research

JoVE Journal

Biology

18.5K 조회수.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

비디오: Using Caco-2 Cells to Study Lipid Transport by the Intestine

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

연구

생물학

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

컴퓨터가 만든 동물 모델 자극

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

의 분리 및 정제 Drosophila</em마그네틱 비드별로 정렬> 주변 뉴런

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

zebrafish의 시각 기능을 연구하기 위해 optokinetic 응답을 사용하여

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

오세영, K와 H, K-ATPase에 의해 양이온 교통 측정<em&gt; Xenopus</em원자 흡수 분광 광도법에 의한&gt; Oocytes : 방사성 동위 원소의 Assays에 대한 대안

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

방법은 3D로 재배 원주민 소장과 카코 2 세포에서 상피 전송 단백질의 기능과 발현을 연구하는

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

장 내분비 세포의 굿 호르몬 분비의 연구 및 라이브 세포 이미징을위한 의도 된 쥐 소장의 혼합 차 문화

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

금 나노 입자와 지질 막 사이의 열 전달을 모니터링하는 테더링 된 바이 레이어 지질 멤브레인

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

시험관 내 대동맥 판막의 석회화를 연구하기 위해 마우스 간막판 세포의 격리

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...