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1、外周血单个核细胞的分离3. 1. 4. 2密度梯度分离从肘静脉无菌采集外周EDTA抗凝血5ml,用等量的生理盐水稀释，将稀释血 液按1： 1比例小心缓慢加在淋巴细胞分离液上，室温800xg离心30rain，离心后 分四层，上层为血浆、血液稀释液和大部分血小板，下层为红细胞和粒细胞，中 间层是淋巴细胞分离液，分离液和血浆交界部位的乳白色混浊液体就是单个核细 胞层，小心吸取该层，加入10. 15ml生理盐水洗涤，室温250xg离心10min，弃上 清；再重复洗涤两次，弃上清。3。1. 4. 3外周血单个核细胞样品RNA的抽提和纯化将分离好外周血单个核细胞样品的迅速加入血1 trizol，冰浴匀浆使
2、细胞样 品充分裂解。转移到一1. 5ml DEPC处理的无菌离心管中，加入400ul氯仿， 剧烈震荡混匀，4度12000rpm离心10min。转移上清至另一离心管中，加入等 体积氯仿：异戊醇（24： 1），剧烈震荡混匀，4度12000rpm离心10rain。转移 上清至另一离心管中，加入等体积氯仿：异戊醇（24： 1），剧烈震荡混匀，4 度12000rpm离心10min。转移上清至另一离心管中，加入等体积异丙醇，震 荡混匀，室温放置2min，4度12000rpm离心10min。轻轻移弃上清，防止吸 去沉淀，加入lml70%乙醇，悬浮沉淀，4度12000rpm离心10min。轻轻移弃 上清，防止
3、吸去沉淀，4度12000rpm离心lmin，用移液器吸干残余酒精，室 温空气干燥，使酒精挥发，加入50ul DEPC处理的灭菌水，溶解RNA。电泳 察看RNA的浓度和完整性。在微量Tube中配制下列反应液，全量50ul全RNA 20ug1 0xDnase I Buffer 5ulDnase I(Rnase-free, 5U / u1) 2ulRnase Inhibitor(40U / u1) 0. 5ulDEP C 处理水 upto 50ul37C反应20. 30分钟。加入50ul的DEPC处理水。加入100ul(等量)的苯酚/ 氯仿/异戊醇(25： 24-1),充分混匀。离心，取上层(水层)
4、移至另一微量离心管中。加入10ul(1 / 10量)的3 M NaOAC(pH5. 2)。加入250ul(2. 5倍量)的冷无水乙醇，.20。C放置30. 60分钟。离心回收沉淀，用70%的冷 乙醇清洗沉淀，真空干燥。用适量的DEPC处理水溶解后，进行Agarose电泳确认是否除去基因组DNA。3. 1. 4. 4 RNA反转录上海交通大学博士学位论文RNA(2u 曲 xulPrimer(oligo dT)lulRNase抑制剂0. 5ul补DEPC水至终体积lOul。轻轻混匀并在微量离心机低速将混合液收集在底部。75C水浴5 min，迅速冰浴2rain。离心收集混合液至管底。每个反应管中顺序
5、加入以下试剂：5RT Buffer 4uldNTP Mix(10 mM each) 1 ulRNase抑制剂 O. 5ulDEPC H20 3. 5 ulMMLV ReverseTranscriptase(1 00 units / u1)1 ul42C空气浴孵育hr, 95C 5min。3. 1. 4. 5 cDNA的荧光定量PCR扩增(20ul体系)荧光定量Master MIX(含SYBR荧光染料)10ulForward Primer l ulReverse Primer 1 ulTemplate 1 ul补DEPC水至终体积20ul。引物序列见表10。肠系膜淋巴结细胞MLNs和固有层单核细
6、胞LPMCs1. 在无菌的条件下分离MLNs,在HBSS液中轻轻压碎为单个细胞悬液。2. 清洗细胞悬液并将细胞重新分散在完全RPMI1640培养液中。(RPMI 1640 containing 10% 胎牛血清,2mmol/L谷氨酰胺,25mmol/L HEPES buffer, and 100 U/mL盘尼西林，100 mg/mL链霉素).自新鲜的结肠组织中分离LPMCs。1. 简而言之，结肠样本用无钙镁离子的HBSS液清洗2. 然后再在含有0.75mM EDTA (Sigma) and 1mM DTT (Sigma)的HBSS中at 37C孵育30 min，从而分离上皮细胞。3. 将组织放
7、于含有400 U/mL胶原酶IV (Sigma) and 0.01mg/mL脱氧核糖 核酸酶DNase I (Sigma)的RPMI中37C震荡孵育，进一步消化。4. 上一步重复2-3次。5. 用40-100%不同梯度的percoll将释放出来的细胞纯化。将LPMCs在培养皿中37C孵育3上 从而分理处粘连细胞，之后，LPMCs中充满T细胞。将106的MLNs和富含T细胞的LPMCs放于含有完全培养基的96孔平板中， 加入或者不加plate-coated鼠抗CD3抗体(8 mg/mL,R&D Systems)和可溶性的抗CD28 抗体(1 mg/mL, R&D Systems)，孵育 48h。
8、2.3. Purification and culture of lamina propria mononuclear cellsLamina propria mononuclear cells (LPMCs) were isolated using a modified dithiothreitol/EDTA and collagenase method as previously described?0 In brief the epithelial layer was removed by agitating the biopsy specimens in 1 mM EDT4. The
9、denuded mucosal sample was then mechanically disrupted and incubated at 37 DC in RPMI 1640 containing 10% fetal calf serum (FCS), 100 pg/ml streptonnycinj 100 UZ ml penicillin, 50 gg/ml gentamicint and 1 mg/ml collagenase V for 90 Eiru The resulting cell fraction was washed three times in RPMI and r
10、esuspended. In all cases sample viability was determined as 90% by Trypan blue staining. For culture experiments the resulting cells were resuspended in RPMI 1640 containing 100 ing/ml streptomycinj 100 U/ml penicillin, 50 jdg/iTil gentamicin 1 mg/ml arnphoterecin and HL-1 (Biowhittaker, Wokingham U
11、K)*, a serum replacement agent.The colonic mucosa was dissected within one hour of resection and lamina propria mononuclear cells were isolated using the DTT-EDTA- collagenase method.7 15 Strips of the mucosa (8-9 g total weight) were washed in Hankss balanced salt solution free of calcium and magne
12、sium (HBSS-CMF) (Flow Lab). They were then washed in HBSS-CMF containing 1 mM of dithiothreitol (DDT) (Sigma Chem) and antibiotics (penicillin 100 U/ml, streptomycin 100 tg/ml, gentamicin 50 ig/ml, and amphotericin 25 ,ug/ml) for 15 minutes at room temperature. After three washings in HBSS- CMF the
13、mucosal strips were chopped into pieces of approximately 3x3 mm. These tiny pieces were then incubated four or five times in HBSS-CMF containing 0 75 mM ethylenediamine tetra-acetic acid (EDTA), 10 mM Hepes buffer, and antibiotics for 45 min at 37C in a humid 5% CO2 atmosphere to remove epithelial c
14、ells. After two washes the pieces were incubated for 10-13 hours at 37C in a humid 5% CO2 atmosphere in complete medium containing 25 U/ml purified collagenase (CLSPA, Worthington).The supernatant was then collected and washed twice in HBSS-CMF and the pellet resuspended in complete medium and layer
15、ed on a Ficoll-Paque density gradient. The resulting lamina propria mononuclear cells were counted and checked for viability using 0a 1% trypan blue (viability ranged from 85-95%). Autologous peripheral blood mononuclear cells were obtained from venous heparinised blood layered on a Ficoll-Paque density gradient. 来自文章Spontaneous release of interferon 7 by intestinal lamina propria lymphocytes in Crohns disease. Kinetics of in vitro response to interferon 7 inducers
