【永生化人類多巴胺能神經元前體細胞（LUHMES）】 ABMGOOD貨號T0284
Abmgood不朽化系列商品 (immortalized cell)
永生化人類多巴胺能神經元前體細胞（LUHMES）- v-myc genes (貨號T0284)
永生化的Dopaminergic Neuronal Precursor Cells人類多巴胺能神經元前體細胞，也稱為Lund Human Mesencephalic隆德人中腦（LUHMES）細胞，是四環素控制的，v-myc過表達的人中腦衍生的細胞系MESC2.10的subclone。 該細胞是獨特的，因為它可以在適當的生長條件下分化為獲得dopaminergic neuron-like多巴胺能神經元樣表型phenotype。 LUHMES在分化後表達功能性dopamine transporter多巴胺轉運蛋白（DAT），vesicular monoamine transporter囊泡單胺轉運蛋白（VMAT-2），tyrosine hydroxylase酪氨酸羥化酶（TH）和β-III tubulin微管蛋白的神經元形式。 除了保留多巴胺能神經元特異性標記DAT, VMAT-2, TH, α-SYN, β-III tubulin之外，LUHMES還表現出電生理學特性，因此使得該細胞系成為neurodevelopmental神經發育研究，疾病建模和neuropharmacology神經藥理學的有意思之神經元細胞Model。
Immortalized Human Dopaminergic Neuronal Precursor Cells (LUHMES) Homo sapiens
永生化的Dopaminergic Neuronal Precursor Cells人類多巴胺能神經元前體細胞，也稱為Lund Human Mesencephalic隆德人中腦（LUHMES）細胞，是四環素控制的，v-myc過表達的人中腦衍生的細胞系MESC2.10的subclone。 該細胞是獨特的，因為它可以在適當的生長條件下分化為獲得dopaminergic neuron-like多巴胺能神經元樣表型phenotype。 LUHMES在分化後表達功能性dopamine transporter多巴胺轉運蛋白（DAT），vesicular monoamine transporter囊泡單胺轉運蛋白（VMAT-2），tyrosine hydroxylase酪氨酸羥化酶（TH）和β-III tubulin微管蛋白的神經元形式。 除了保留多巴胺能神經元特異性標記DAT, VMAT-2, TH, α-SYN, β-III tubulin之外，LUHMES還表現出電生理學特性，因此使得該細胞系成為neurodevelopmental神經發育研究，疾病建模和neuropharmacology神經藥理學的有意思之神經元細胞Model。
| Organism | Homo sapiens |
| Source Organ | 8-week-old fetal human ventral mesencephalon |
| Donor Age | 8 week |
| Donor Gender | undetermined |
| BioSafety Level | II |
| Growth Properties | Adherent |
| Morphology | Flattened|Dendritic processes |
| Recommended Seeding Density | 50,000 – 100,000 cells/cm2; cells will have viability of around 40-50%. Post thawing, it will need 4-5 days for cells to recover in culture; Recommended split ratio is 1:4 to 1:5 |
| Population Doubling | 30 – 40 hours |
| Immortalization Method | Conditional immortalization by tetracyclin-controlled transduction with retrovirus carrying v-myc genes |
| Description | The Immortalized Human Dopaminergic Neuronal Precursor Cells, also known as the Lund Human Mesencephalic (LUHMES) cell line, is a subclone of the tetracycline-controlled, v-myc-overexpressing human mesencephalic-derived cell line MESC2.10. This cell line is unique in that it can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. LUHMES expresses functional dopamine transporter (DAT), vesicular monoamine transporter (VMAT-2), tyrosine hydroxylase (TH) and the neuronal form of β-III tubulin after differentiation. In addition to retaining dopaminergic neuronal-specific markers, LUHMES also exhibit electrophysiological properties, thus making this cell line a valuable neuronal model for neurodevelopmental studies, disease modelling and neuropharmacology. |
| Applications | For Research Use Only |
| Markers | DAT, VMAT-2, TH, α-SYN, β-III tubulin |
永生化人類多巴胺能神經元前體細胞- v-myc genes 細胞特性: Adherent | 不杇方法: tetracyclin-controlled transduction with retrovirus carrying v-myc genes | 來源8-week-old fetal human ventral mesencephalon。細胞型態: Flattened|Dendritic processes。細胞培養液更換大約1週2-3次；細胞繼代為胰蛋白酶溶液0.25% trypsin(TM050)。細胞建議之生長密度為50,000 – 100,000 cells/cm2,; Population Doubling30 – 40 hours細胞的活力約為40-50％。 解凍後，細胞需要4-5天才能恢復培養; 推薦的Seeding比例是1：4至1：5。 細胞培養皿pre-coated需預包覆50μg/ ml poly-L-ornithine (PLO)（TM062）和1μg/ ml fibronectin（EMD Millipore; Cat.FC010）/H 2 O, 細胞37℃下培養至少3小時。該細胞系的基礎培養基是Advanced DMEM / F12（Gibco; 12634010）。complete growth medium完整的生長培養基製備，將下列組分添加到基礎培養基中：1x N2 supplement（ThermoFisher Scientific），2mM L-glutamine（G275）和40ng / ml recombinant bFGF（Z101455）。二氧化碳（CO2）：5％，溫度：37.0℃。每2-3天更換一次complete media。不要讓complete media顏色變成橙黃色。
當受到壓力時，細胞會形成clumps圓團而不是monolayer單層。建議細胞生長至50-60％的細胞密度時，繼代培養。為了繼代培養細胞，使用0.25％胰蛋白酶-EDTA（TM051）。加入胰蛋白酶後，將細胞在室溫下孵育2-3分鐘並攪拌培養皿直至90％的細胞脫落。使用胰蛋白酶中和溶液（Lonza，貨號CC-5002）立即中和胰蛋白酶。添加胰蛋白酶中和溶液兩倍的胰蛋白酶使用量。 200×g離心2-3分鐘。吸出上清液，並在完全培養基中resuspend細胞。Plate細胞到預熱的PLO-fibronectin-coated vessels在37℃。如果細胞出現壓力，請避免繼代培養。注意：如果在周末（或超過2天）離開細胞，請確保做到高比例之細胞密度培養（1：4至1：5）。
為了將細胞分化成神經元，在細胞生長至40-50％的密度後將培養基更換為分化培養基。為了製備完全的分化培養基，將以下組分添加到基礎的Advanced DMEM / F12（Gibco; 12634010）中：1×N2補充物，2mM L-谷氨酰胺，1mM dbcAMP，1μg/ ml四環素和2ng / ml重組人GDNF（Z101055）。在測試神經元特異性標記前，讓細胞在分化培養基中靜置4-6天。
HOT!! 黴漿菌PCR偵測 | DAPI染核 | 細胞內黴漿菌去除劑 | 水浴槽抑菌劑 | 噴霧式去黴劑 等系列商品
Abmgood不朽化系列商品 (immortalized reagenet) Ready-to-use
Abmgood公司於人類與老鼠不朽之細胞株上，具有幾十年的實驗經驗；提供一系列不朽 (immortalized)化之細胞株、及不朽化之試劑商品。ready-to-use定量好之retrovirus、lentiviru、 adenovirus，可直接加於細胞中進行感染表現；病毒液包括有SV40重組病毒、EBV重組病毒、hTERT病毒液、Myc基因病毒液、p53基因病毒液、Rb基因病毒液、Ras基因病毒液。提供106及109兩種包裝力價之病毒液；亦提供一系列病毒力價濃縮與定量試劑商品。
細胞不朽化的工具中，SV40 T antigen是最簡單且最常用來造成不朽化細胞生成的方法。多數的病毒基因和抑癌基因（p53、Rb等）的不活化相關，會誘導細胞處於replicative senescence的階段。目前也有研究顯示被感染的細胞，SV40 T anbtigen會誘導端粒酶（Telomerase）的活性。與端粒（telomere）長度相關的Telomerase Reverse Transcriptase protein (TERT)是一種不朽細胞的研究應用。平常細胞中，hTERT是處於不活化態，必要時會進行活化保持telomere的長度，避免細胞發生replicative senescence的現象。某些細胞hTERT 過度表現（over-expression）對於誘發細胞不朽化是無效的（如初代上皮細胞），反而會造成細胞死亡；同時表現hTERT catalytic subunit以及p53或RB siRNA在人類初代卵巢上皮細胞會造成細胞不朽化。過度表現Ras或Myc T58A mutants也有初代細胞不朽化的現象產生。
引用文獻-永生化人類多巴胺能神經元前體細胞（LUHMES）- v-myc genes (貨號T0284)
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