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胰蛋白酶-EDTA溶液(0.25%:0.02%,含酚红)

一键复制产品信息

货号:AWC0239

价格: ¥79

规格: 100ml 500ml

  • 产品概述
  • 胰蛋白酶-EDTA溶液(0.25%:0.02%,含酚红)

    产品简介:

    胰蛋白酶(Trypsin)是由胰脏产生没有活性的胰蛋白酶原分泌到小肠后,小肠内的肠肽酶会活化该酶原,形成胰蛋白酶。胰蛋白酶的特点在于已经活化的胰蛋白酶,能够继续活化更多胰蛋白酶原,这种过程即自动催化。胰蛋白酶在小肠工作,它会将蛋白质水解为肽,进而分解为氨基酸,其最适温度约为37℃。

    Trypsin-EDTA Solution(0.25%:0.02%,含酚红)由0.25%胰酶、0.02%EDTA、少量酚红等组成,经过滤除菌。本试剂可以直接用于培养细胞的消化,或者一些组织的消化,通常室温下1min左右就可以消化下大多数贴壁细胞。

    自备材料:

    1、 PBS、Hanks液或无血清培养液

    2、 显微镜

    3、 离心机

    操作步骤(仅供参考):

    1、 贴壁细胞的消化

    ① 吸除培养液,用无菌PBS、Hanks液或无血清培养液洗涤细胞1次,以去除残余的血清。

    ② 加入少量Trypsin-EDTA Solution,略盖过细胞即可,室温放置0.5~2min,不同的细胞消化时间有所不同。

    ③ 显微镜下观察,细胞明显收缩,并且肉眼观察培养皿底部发现细胞的形态发生明显的变化;或者用枪吹打细胞发现细胞刚好可以被吹打下来,吸除胰酶细胞消化液。加入含血清的完全细胞培养液,吹打下细胞,即可直接用于后续实验。

    ④ 如果发现消化不足,则加入Trypsin-EDTA Solution重新消化。

    ⑤ 如果发现细胞消化时间过长,未及吹打细胞,细胞已经有部分直接从培养器皿底部脱落,直接用胰酶细胞养液把细胞全部吹打下来。1000~2000g离心1min,沉淀细胞,尽量去除胰酶细胞消化液后,加入含血清的完全培养液重新悬浮细胞,即可用于后续实验。

    2、 组织的消化

    ① 不同的组织需要消化的时间相差很大,通常以消化后可以充分打散组织为宜。

    注意事项:

    1、 尽量减少反复冻融的次数,以免失效。

    2、 在使用Trypsin-EDTA Solution过程中,要特别注意避免消化液被细菌污染。

    3、 Trypsin-EDTA Solution消化细胞时间不宜过长,否则细胞铺板后生长状况会较差。

    4、 为了您的安全和健康,请穿好实验服并佩戴一次性手套和口罩操作。

    5、 本产品仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。

    产品组成

    名称

    货号

    规格

    storage

    胰蛋白酶-EDTA溶液(0.25%∶0.02%,含酚红)

    AWC0239a

    100ml

    -20℃

    胰蛋白酶-EDTA溶液(0.25%∶0.02%,含酚红)

    AWC0239b

    500ml

    -20℃

    注意:

    1.本产品仅供科研使用。请勿用于医药、临床诊断或治疗。食品及化妆品等用途。请勿存放于普通住宅区。

    2.为了您的安全和健康,请穿好实验服并佩戴一次性手套和口罩操作。

    3.实验结果可由多种因素影响,相关处理只限于产品本身,不涉及其他赔偿。


    参考文献 (3)

    JOURNAL OF ANIMAL SCIENCE IF:3.3

    The present study aimed to investigate the effects of lipopolysaccharide (LPS) stimulation on oxidative damage, apoptosis, and glutamine (Gln) transporter Alanine-Serine-Cysteine transporter 2 (ASCT2) expression in porcine small intestinal epithelial cells (IPEC-J2), and preliminarily elucidated the relationship between ASCT2 expression level and oxidative damage and apoptosis of IPEC-J2 cells. IPEC-J2 cells were treated without (control group, CON, N = 6) or with 1 μg/mL LPS (LPS group, LPS, N = 6). Cell viability, lactate dehydrogenase (LDH) content, malonaldehyde (MDA), anti-oxidant enzymes (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GSH-Px], and total anti-oxidant capacity [T-AOC]), apoptosis of IPEC-J2 cells, the expression of Caspase3, the expression of ASCT2 mRNA and ASCT2 protein was detected. The results showed that LPS stimulation of IPEC-J2 cells significantly reduced the cell viability, and anti-oxidant enzymes activity (SOD, CAT, and GSH-Px), and significantly increased LDH and MDA release. Flow cytometry results showed that LPS stimulation significantly increased the late apoptosis rate and the total apoptosis rate of IPEC-J2 cells. The immunofluorescence results showed that the fluorescence intensity of LPS stimulated IPEC-J2 cells was significantly enhanced. LPS stimulation significantly decreased the mRNA and protein expression of ASCT2 in IPEC-J2 cells. The correlation analysis showed that ASCT2 expression was negatively correlated with apoptosis, and positively correlated with the anti-oxidant capacity of IPEC-J2 cells. According to the results of this study, it can be preliminarily concluded that LPS promotes the apoptosis and oxidative injury of IPEC-J2 cells by down-regulating the expression of ASCT2.

    CELL BIOCHEMISTRY AND FUNCTION IF:2.7

    SENP3, a member of the sentrin-specific protease family, plays a pivotal role in lipid metabolism and the pathogenesis of fatty liver disease by regulating the dynamic process of SUMOylation. It has previously been demonstrated that SREBP2 is SUMOylated. However, the function and regulatory mechanism of SENP3-mediated SREBP2 de-SUMOylation in hepatocyte steatosis is unclear. Bioinformatic analysis (proteomes from whole cell extract and nuclear extraction, integrated transcriptomes, RNA sequence) SENP3-driven SREBP2 de-SUMOylation in lipid metabolism; Oil red O, nuclear and cytoplasmic extraction, western blot and immunofluorescence suggest SENP3 vitally contributes to hepatocyte steatosis; NLS predictions, CO-IP, co-transfection of plasmids, confocal microscopy indicates nuclear SENP3 associates with SREBP2 to promote its nuclear translocation; molecular docking and mutation assay confirmed SENP3 is responsible for de-SUMOylation SREBP2 at R576. Treatment with OA increased the level of both SENP3 and its nuclear fraction in HepG2 cells. Interfering with the SUMOylation and deSUMOylation cycle induced hepatocyte steatosis by overexpressing SENP3 and using a SUMO inhibitor, GA. Mechanistically, it is observed that the co-localization between SREBP2 and SENP3 is increased. SENP3 is responsible for de-SUMOylation SREBP2 at R576. Moreover, SENP3-mediated de-SUMOylation may also decrease ZMIZ1-ligated SUMO3 binding to SREBP2 at K464 in the nucleus. RNA interference of SREBP2 cannot reverse SENP3-overexpressed cell steatosis under fatty acid treatment. Expression of SREBP2 in vitro could upregulate the nuclear locations of CCTα binding to DNA without altering the active forms. Our findings demonstrate that de-SUMOylation is an important regulatory mechanism that governs the lipid accumulation of SREBP2 in mammalian cells. Also, the critical role of the de-SUMOylation of SREBP2 by SENP3 to exacerbate steatosis may be a potential therapeutic target for metabolic diseases like MAFLD/MASH. Summary Previous studies have reported SUMOylation of SREBP2, and our published works have centered on SUMOylation of nuclear proteins and hepatocyte metabolism. However, why the K464R mutation completely abolishes SUMO3 modification of SREBP2 remains unsolved. SENP3 de-SUMOylated SREBP2 at R576 to promote its nuclear translocation by de-SUMOylation of SREBP2. This site also influences CCT association with the nuclear envelope and DNA. Building on this, we have filled these gaps and extended the field by demonstrating that SREBP2 recruits CCT dimers to DNA without altering the nuclear size. Our goal is to elucidate the nuclear regulatory mechanisms of metabolic enzymes, providing insights for precision targeting and clinical translation of metabolic diseases like MAFLD.

    JOURNAL OF ANIMAL SCIENCE IF:2.7

    The present study aimed to investigate the effects of deoxynivalenol (DON) stimulation on inflammatory injury and the expression of the glucose transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter protein 2 (GLU2) in porcine small intestinal epithelial cells (IPEC-J2). Additionally, the study aimed to provide initial insights into the connection between the expression of glucose transporters and the inflammatory injury of IPEC-J2 cells. DON concentration and DON treatment time were determined using the CCK‑8 assay. Accordingly, 1.0 µg/mL DON and treat for 24 h was chosen for subsequent experiments. Then IPEC-J2 cells were treated without DON (CON, N =6) or with 1 μg/mL DON (DON, N=6). Lactate dehydrogenase (LDH) content, apoptosis rate, and proinflammatory cytokines including interleukin (IL)-1β, Il-6 and tumor necrosis factor α (TNF-α) were measured. Additionally, the expression of AMP-activated protein kinase α1 (AMPK-α1), the content of glucose, intestinal alkaline phosphatase (AKP) and sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) activity, the expression of SGLT1 and GLU2 of IPEC-J2 cells were also analyzed. The results showed that DON exposure significantly increased LDH release and apoptosis rate of IPEC-J2 cells. Stimulation with DON resulted in significant cellular inflammatory damage, as evidenced by a significant increase in proinflammatory cytokines (IL-1β, IL-6 and TNF-α). Additionally, DON caused damage to the glucose absorption capacity of IPEC-J2 cells, indicated by decreased levels of glucose content, AKP activity, Na+/K+-ATPase activity, AMPK-α1 protein expression, and SGLT1 expression. Correlation analysis revealed that glucose absorption capacity was negatively correlated with cell inflammatory cytokines. Based on the findings of this study, it can be preliminarily concluded that the cell inflammatory damage caused by DON may be associated with the decreased glucose absorption.

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