DMEM基础培养基

实验要点及说明：

1．本方法适用于贴壁细胞培养，而不适用于悬浮细胞培养，悬浮细胞可使用滴片法； 

2．所使用的盖玻片应该为玻璃**，并经过铬酸洗液处理； 

3．盖玻片非常薄，易碎，取放盖玻片时动作要轻； 

4．如果需要更多生长状态一致的细胞，可以使用较大的培养皿，但不宜过大，以避免培养液的浪费和增加污染机率； 

5．如果细胞贴壁生长能力较差，可将盖玻片在0.5%多聚赖氨酸溶液中浸泡5-10分钟并自然晾干。

培养方法

传代方法 将旧培养液吸除，PBS清洗两遍后，加入6mL（/100mm皿）胰酶，在显微镜下观察，期间禁止摇晃培养皿，细胞刚有脱落时，则吸除大部分胰酶，留约0.5mL，移至培养箱消化，约2min取出。传代用12mL CM1-1培养液终止消化，轻轻吹打均匀细胞，后可分3~6皿培养；

生长条件 37℃，5%CO2，CM1-1培养液。CM1-1培养液：90%DMEM-H+10%FBS。DMEM-H：DMEM高糖培养液，含谷氨酰胺，含丙酮酸钠。

存储条件 冻存则用6mL冻存液（90%FBS+10%DMSO）终止消化，吹打均匀，分为6支冻存管，用程序降温盒于-80℃冻存，过夜转移至液氮中保存。

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培养操作步骤 ：

1．用盖片镊将盖玻片自75%乙醇中取出，用无菌丝绸布擦拭干净，不要用纱布； 

2．将盖玻片轻轻放入6孔培养板（每孔一片）或培养皿中（每个平皿可放置2-3片）； 

3．在距离紫外灯直射范围内20-30 厘米处照射2-3小时； 

4．将经过计数的细胞悬浮液移入培养板中，使盖玻片完全浸在培养液中； 

5．将培养板在5% CO2水浴孵箱中37℃孵育2-3天，当贴壁细胞生长至覆盖培养板底部2/3面积时，将培养板取出，用盖片镊轻轻取出盖玻片，用蒸馏水漂洗后即可进行快速固定以及细胞化学检测。 

细胞培养方法：

1、细胞传代：细胞密度达到80-90%时即可传代

①弃去培养上清，用PBS或生理盐水清洗1-2次；

②加入2ml0.25%胰酶（T25瓶），使胰酶覆盖整个瓶或皿，盖好放入培养箱消化；

③1-2min后，显微镜下观察细胞，若大部分细胞回缩且有少量细胞脱落，轻轻吹打下确认消化情况后加入完全培养基终止消化；若细胞还是贴壁，放回培养箱继续消化至可以轻轻吹打下为止；

④将细胞悬液1000RPM左右条件下离心4min，弃上清；

⑤用新鲜培养基重悬后加入培养瓶或皿中，T25培养瓶加6-8ml培养基；

⑥悬浮细胞直接离心收集，细胞沉淀重悬后分到新培养瓶中。

2、细胞复苏：

①将冻存管在37℃温水中快速摇晃融化，时间1min左右，加入4-5ml培养基混匀。

②在1000RPM左右条件下离心4min，弃上清,加1-2ml培养基吹匀，将细胞悬液加入培养瓶中，补加适量培养基。

3、细胞冻存：待细胞生长状态良好时进行细胞冻存保种

①弃去培养上清，用PBS或生理盐水清洗1-2次，加入1mL 0.25%胰蛋白酶（T25瓶）

②1-2min后，显微镜下观察细胞，大部分细胞回缩且有少量细胞脱落，轻轻吹打下确认消化情况后加入完全培养基终止消化；

③将细胞悬液1000RPM左右条件下离心4min，弃上清，加1ml冻存液重悬细胞；

④将冻存管放入程序降温盒，放入-80℃冰箱，4小时后将冻存管转入液氮罐储存。

Subunit : Interacts with homodimeric PDGFA, PDGFB and PDGFC, and with heterodimers formed by PDGFA and PDGFB. Monomer in the absence of bound ligand. Interaction with dimeric PDGFA, PDGFB and/or PDGFC leads to receptor dimerization, where both PDGFRA homodimers and heterodimers with PDGFRB are observed. Interacts (tyrosine phosphorylated) with SHB (via SH2 domain). Interacts (tyrosine phosphorylated) with SHF (via SH2 domain). Interacts (tyrosine phosphorylated) with SRC (via SH2 domain). Interacts (tyrosine phosphorylated) with PIK3R1. Interacts (tyrosine phosphorylated) with PLCG1 (via SH2 domain). Interacts (tyrosine phosphorylated) with CRK, GRB2 and GRB7. Interacts with human cytomegalovirus/HHV-5 envelop glycoprotein B/gB.

Subcellular Location : Cell membrane; Single-pass type I membrane protein. Note=The activated receptor is rapidly internalized and degraded.

Tissue Specificity : Detected in platelets (at protein level). Widely expressed. Detected in brain, fibroblasts, smooth muscle, heart, and embryo. Expressed in primary and metastatic colon tumors and in normal colon tissue.

Post-translational modifications : N-glycosylated.

Ubiquitinated, leading to its degradation (Probable).

Autophosphorylated on tyrosine residues upon ligand binding. Autophosphorylation occurs in trans, i.e. one subunit of the dimeric receptor phosphorylates tyrosine residues on the other subunit. Phosphorylation at Tyr-731 and Tyr-742 is important for interaction with PIK3R1. Phosphorylation at Tyr-720 and Tyr-754 is important for interaction with PTPN11. Phosphorylation at Tyr-762 is important for interaction with CRK. Phosphorylation at Tyr-572 and Tyr-574 is important for interaction with SRC and SRC family members. Phosphorylation at Tyr-988 and Tyr-1018 is important for interaction with PLCG1.

DISEASE : Note=A chromosomal aberration involving PDGFRA is found in some cases of hypereosinophilic syndrome. Interstitial chromosomal deletion del(4)(q12q12) causes the fusion of FIP1L1 and PDGFRA (FIP1L1-PDGFRA). Mutations that cause overexpression and/or constitutive activation of PDGFRA may be a cause of hypereosinophilic syndrome.

Defects in PDGFRA are a cause of gastrointestinal stromal tumor (GIST) [MIM:606764]. Note=Mutations that cause constitutive activation of PDGFRA may be a cause of gastrointestinal stromal tumor (GIST).

Similarity : Belongs to the protein kinase superfamily. Tyr protein kinase family. CSF-1/PDGF receptor subfamily.

Contains 5 Ig-like C2-type (immunoglobulin-like) domains.

Contains 1 protein kinase domain.

Database links : UniProtKB/Swiss-Prot: P16234.1

PDGFR-α是膜受体，具有酪氨酸酶的活性，与其配体PDGF结合后激活与细胞增殖有关的酶及基因。 PDGFR亦表达于上皮、内皮细胞，前列腺、皮肤、肾小球等上皮细胞均有PDGFR表达. 亦有学者报道血小板源性生长因子受体α抗体在细胞胞浆胞膜、胞核都有不同的表达还有人认为：PDGF及其受体一般表达于浸润病变组织的炎症细胞附近，组织缺血损伤、肾小球高压、因素作用、炎症细胞浸润、实质细胞活化等导致PDGFR表达增强。

英文名称  Anti-Phospho-PDGFRA(Tyr572)/PDGFRB(Tyr579)  

中文名称  磷酸化血小板源性生长因子受体α

别    名  PDGFRA(phospho Y572); PDGF Receptor alpha(Tyr849)/PDGF Receptor beta (Tyr857); Platelet–dirived growth factor receptor-alpha; Alpha platelet derived growth factor receptor; CD 140a; CD140a; CD140a antigen; MGC74795; PDGF alpha chain; PDGF R alpha; PDGFR 2; PDGFR A; PDGFR alpha; PDGFR2; PDGFRA; Platelet derived growth factor receptor 2; Platelet derived growth factor receptor alpha; Platelet derived growth factor receptor alpha polypeptide; PGFRA_HUMAN.

浓    度  1mg/1ml

规 格  0.1ml/100μg
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