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胰蛋白酶-EDTA溶液(0.25%:0.02%)

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货号:AWC0238

价格: ¥70

规格: 100ml 500ml

  • 产品概述
  • 胰蛋白酶-EDTA溶液(0.25%:0.02%)

    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%)由0.25%胰酶、0.02%EDTA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。

    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 (7)

    Cell Death & Disease IF:9.6

    Ovarian cancer (OC) is often detected at an advanced stage and has a high recurrence rate after surgery or chemotherapy. Thus, it is essential to develop new strategies for OC treatment. This study tended to investigate the effects of endothelial cell-specific molecule 1 (ESM1) in OC. The impact of ESM1 on lipid metabolism was investigated through the regulation of ESM1 expression. Differential genes regulated by ESM1 were screened by mRNA sequencing. The role of autophagy in ESM1 regulation on lipid metabolism was explored using autophagy inhibitor chloroquine (CQ). Co-IP, dual-luciferase reporter assay, actinomycin D treatment assay, and others were used to analyze the mechanism of ESM1 regulation on lipid metabolism. The xenograft mouse model was constructed to explore the impact of ESM1 regulation on OC development. The regulatory mechanism of ESM1 in OC patient samples was verified by using microarray analysis and the Log-rank (Mantel-Cox) test. After ESM1 silencing, cholesterol synthesis decreased and lipolysis increased. mRNA sequencing revealed that ESM1 regulation on lipid metabolism was related to Beclin 1 (BECN1). In vitro experiments, ESM1 inhibited lipolysis by suppressing BECN1-mediated autophagy. BECN1 expression was regulated by the transcription factor Kruppel-like factor 10 (KLF10). The competitive binding between BECN1 and HSPA5 promoted the ubiquitination degradation of HMGCR, thereby inhibiting cholesterol production. The intervention experiment with exogenous cholesterol showed a positive correlation between m6A reader IGF2BP3 expression and cholesterol content. Mechanistically, IGF2BP3 regulated the stability of ESM1 mRNA. In vivo experiments, ESM1 modified by m6A methylation promoted cholesterol synthesis and inhibited lipolysis. High expression of ESM1 predicted poor prognosis in OC patients. ESM1 regulated lipid metabolism through IGF2BP3/ESM1/KLF10/BECN1 positive feedback, which was a promising target for OC treatment.

    PHYTOMEDICINE IF:6.7

    Background Quercetin, the key ingredient in Xiaoyao Kangai Jieyu Formula, has been previously found to relieve breast cancer-related depression (BCRD). Purpose We want to explore the potential mechanisms and therapeutic targets of quercetin alleviating BCRD. Methods BALB/c mice were injected subcutaneously with 4T1 cells and corticosterone (CORT) to create a BCRD mice model. The primary hippocampal neurons were co-induced with 10 μg/mL lipopolysaccharide (LPS) and 200 μM CORT for 6 h to establish an in vitro model of BCRD. Quercetin was applied to explore its effect on disease symptoms, gut microbiota, and lipid metabolism of BCRD mice. Lipid metabolism-related genes were screened based on network pharmacology. Molecular docking was employed to prove whether quercetin bound to prostaglandin-endoperoxide synthase 2 (PTGS2). PTGS2 overexpression was carried out to explore the underlying mechanism of quercetin treatment on BCRD. Results Quercetin treatment not only altered the composition and abundance of gut microbiota but also alleviated abnormal lipid metabolism in BCRD mice. In particular, quercetin down-regulated BCRD and lipid metabolism-related genes screened by network pharmacology, especially PTGS2. Further, molecular docking verified the stable binding between quercetin and PTGS2. In hippocampal neurons, quercetin promoted proliferation but reduced ferroptosis-related markers (total Fe, Fe2+, MDA, and ROS) levels by targeting PTGS2. In BCRD mice, quercetin reduced the high immobility time and increased the sucrose preference rate and serotonin (5-HT), dopamine (DA), and noradrenaline (NE) levels. Meanwhile, quercetin increased CD4+/CD8+ T cells ratio and IL-2 and IFN-γ levels but reduced CA153 and IL-10 levels to alleviate BCRD development. However, PTGS2 overexpression reversed these effects of quercetin on BCRD. Conclusion Quercetin inhibited neuronal ferroptosis and promoted immune responses in BCRD mice by targeting the lipid metabolism-related gene PTGS2. This provided a reference for quercetin in the treatment of BCRD.

    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.

    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY IF:3.1

    Long non-coding RNAs (Lnc RNAs) are proven to participate in liver cancer (LC) regulation. The regulation of miR-21 by lnc NBAT1 has been studied in other cancers. However, the effect of this regulation on LC and its specific mechanism remains unclear. Lnc NBAT1 and miR-21 expressions in clinical tissues were measured by RT-qPCR. PDCD4, AP-1, p–c-Fos, p–c-Jun, and cyclin D1 expressions were analyzed by Western blot. Overexpression of lnc NBAT1 was studied to explore its influence on malignant behaviors of Bel7402 cells and the development of LC in the xenograft mouse model (XMM). The regulation mechanism of lnc NBAT1 in LC was explored by lnc NBAT1 overexpression, miR-21 mimic treatment, or PDCD4 silencing in Bel7402 cells. Lnc NBAT1 expression was downregulated while miR-21 expression was upregulated in LC tissues and cell lines. In comparison with LX-2 cells, the expressions of PDCD4 and AP-1 were downregulated in Bel7402 cells, while those of p–c-Fos, p–c-Jun, and cyclin D1 were upregulated. Further, lnc NBAT1 was found to localize primarily in the cytoplasm of Bel7402 cells. Overexpression of lnc NBAT1 enhanced cell apoptosis, blocked the cell cycle, suppressed malignant behaviors of Bel7402 cells, and inhibited tumor progression in the XMM. Mechanistically, lnc NBAT1 functioned as a competing endogenous RNA (ceRNA) by binding to the downstream target miR-21 to stabilize the expressions of PDCD4 and AP-1, thereby inhibiting malignant behaviors of Bel7402 cells. Lnc NBAT1 suppressed malignant behaviors of LC cells through the miR-21/PDCD4/AP-1 axis. Lnc NBAT1 might be a promising biomarker for LC treatment.

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS IF:3

    Objective Stem cell therapy is expected to become a new treatment for central nervous system damage associated with perinatal hypoxic-ischemic encephalopathy (HIE), but the specific effects are unknown. This study explores the effects of human umbilical cord blood (HUCB) cells-secreted exosomal (HUCB-ex) MFG-E8 in neonatal rats with hypoxic-ischemic brain damage (HIBD), aiming to gain a theoretical foundation for the cure of perinatal HIE. Methods HIBD model was constructed in the Sprague Dawley rats (7-day-old). Rats were then intervened with 1×10 6 HUCB cells, HUCB-ex, or HUCB-ex oe-MFG-E8 , and HUCB-ex si-MFG-E8 . Primary microglia from rats were induced with oxygen-glucose deprivation and re-oxygenation (OGD/R), then co-cultured with either HUCB or primary neuronal cells, and subjected to treatment with HUCB-ex, HUCB-ex oe-MFG-E8 , HUCB-ex si-MFG-E8 , or Stattic. The expression of polarization factors and secreted factors in the microglia was measured using RT-qPCR, immunofluorescence, and western blot. Neuronal cell damage was assessed using MTT assays and flow cytometry. Behavioral impairments and brain tissue damage in the rats were evaluated using assays including the geotaxis reflex, cliff avoidance response, grip strength test, hematoxylin-eosin staining, TTC staining, and immunofluorescence. Results Early intervention with HUCB cells in HIBD rats increased test scores, decreased brain tissue weight, infarct area, as well as the IL-6, TNF-α, and IL-1β levels, and increased MFG-E8 levels. HUCB cells also decreased the levels of CD11b/c + CD45hi cells in HIBD rat brain tissue, and increased the levels of CD206 + CD11b/c + cells. In vitro experiments confirmed high expression of MFG-E8 in HUCB-ex. HUCB-ex si-MFG-E8 inhibited M2 polarization and induced neuronal cell injury through the SOCS3/STAT3 pathway. HUCB-ex and HUCB cells have equivalent therapeutic effects in HIBD rats. The treatment effectiveness of HUCB-ex was improved after delivering HUCB-ex oe-MFG-E8 , while was blocked after delivering HUCB-ex si-MFG-E8 . Conclusions HUCB-ex oe-MFG-E8 promoted M2 polarization of microglial and inhibited neuronal cell apoptosis through the SOCS3/STAT3 pathway, to alleviate behavioral disorders and brain tissue damage in HIBD rats.

    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.

    COMBINATORIAL CHEMISTRY & HIGH THROUGHPUT SCREENING IF:1.6

    Background: Lung adenocarcinoma (LUAD) is one of the most common malignant cancers. Neutrophil extracellular traps (NETs) have been discovered to play a crucial role in the pathogenesis of LUAD. We aimed to establish an innovative prognostic model for LUAD based on the distinct expression patterns of NETs-related genes. Methods: The TCGA LUAD dataset was utilized as the training set, while GSE31210, GSE37745, and GSE50081 were undertaken as the verification sets. The patients were grouped into clusters based on the expression signature of NETs-related genes. Differentially expressed genes between clusters were identified through the utilization of the random forest and LASSO algorithms. The NETs score model for LUAD prognosis was developed by multiplying the expression levels of specific genes with their corresponding LASSO coefficients and then summing them. The validity of the model was confirmed by analysis of the survival curves and ROC curves. Additionally, immune infiltration, GSEA, mutation analysis, and drug analysis were conducted. Silencing ABCC2 in A549 cells was achieved to investigate its effect. Results: We identified six novel NETs-related genes, namely UPK1B, SFTA3, GGTLC1, SCGB3A1, ABCC2, and NTS, and developed a NETs score signature, which exhibited a significant correlation with the clinicopathological and immune traits of the LUAD patients. High-risk patients showed inhibition of immune-related processes. Mutation patterns exhibited variability among the different groups. AZD3759, lapatinib, and dasatinib have been identified as potential candidates for LUAD treatment. Moreover, the downregulation of ABCC2 resulted in the induction of apoptosis and suppression of migration and invasion in A549 cells. Conclusion: Altogether, this study has identified a novel NET-score signature based on six novel NET-related genes to predict the prognosis of LUAD and ABCC2 and has also explored a new method for personalized chemo-/immuno-therapy of LUAD.

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