Cell-Tracker Green CMFDA  活細(xì)胞示蹤探針（綠色）

重要提醒（購買或初次使用前）請(qǐng)務(wù)必查閱：

產(chǎn)品關(guān)鍵詞：

Cell-Tracker Green CMFDA；Cell-Tracker Red CMTPX；Cell movement 細(xì)胞運(yùn)動(dòng)；Cell location 細(xì)胞定位；Cell Tracker dye 細(xì)胞示蹤探針；CAS NO：136832-63-8；

訂購信息：

產(chǎn)品描述

Cell-Tracker熒光探針是用來監(jiān)測(cè)細(xì)胞運(yùn)動(dòng)、定位、增殖、遷移、趨化和侵襲的優(yōu)秀工具。

Cell-Tracker Green CMFDA，英文全名：5-chloromethylfluorescein diacetate，能自由穿透細(xì)胞膜進(jìn)入細(xì)胞，在胞內(nèi)轉(zhuǎn)化生成不具細(xì)胞膜滲透性的反應(yīng)產(chǎn)物。該產(chǎn)物幾次傳代都能良好的保留在活細(xì)胞。在細(xì)胞群內(nèi)，染料只會(huì)轉(zhuǎn)移到子代細(xì)胞，不會(huì)轉(zhuǎn)移到鄰近細(xì)胞。

Cell-Tracker Red CMTPX特地設(shè)計(jì)使其至少72h（典型有3~6代）能展示熒光，此染料表現(xiàn)出理想的示蹤特征：穩(wěn)定、工作濃度下無毒性、良好保留在細(xì)胞，且在生理pH下呈明亮熒光。另外，Cell-Tracker Red CMTPX的激發(fā)和發(fā)射光譜（492/517nm）與紅色熒光蛋白R(shí)FP很好的分開，適用于多重標(biāo)記（見附表1. Cell-Tracker熒光探針的光譜特征）。

產(chǎn)品特性

1）  CAS NO.：136832-63-8

2）  同義名：CellTracker™ Green CMFDA; Spiro(isobenzofuran-1(3H),9'-(9H)xanthen)-3-one, 3',6'-bis(acetyloxy)-5-(chloromethyl)-;

3）  分子式：C25H17ClO7

4）  分子量：464.86 g/mol

5）  外觀：無色固體

6）  溶解性：溶于DMSO

7）  化學(xué)結(jié)構(gòu)式： 

保存與運(yùn)輸方法

保存：-20℃避光干燥保存，至少2年有效。

運(yùn)輸：冰袋運(yùn)輸。

注意事項(xiàng)

1. 熒光染料都存在淬滅的問題，保存和操作過程中注意避光。

2. 避免使用含氨基和巰基的緩沖液。

3. 為了您的安全和健康，請(qǐng)穿實(shí)驗(yàn)服并戴一次性手套操作。

應(yīng)用示例

文獻(xiàn)1）Nasir NAM et al.Fluorescent cell tracer dye permits real‐time assessment of re‐epithelialization in a serum‐free ex vivo human skin wound assay. Wound Repair Regen. 2019 Jan;27(1):126-133.

Method (Cell tracker dye uptake within human ex vivo wounds in situ)：Four microliters of 25 μM CMFDA (or CMTPX) was added to the wound surface for 30 minutes, then washed dropwise with phosphate buffered saline.For cell viability studies, 1 μg/mL of propidium iodide (PI) was cotreated alongside CMFDA. Planimetric imaging was conducted using a Leica M205 FA upright Stereomicroscope using a 5x/0.50 pLANapo LWD objective at the equivalent of 64x magnification and captured using a DFC 565FX camera through LAS AF v3.1.0.8587 software. Specific band‐pass filter set for green fluorescent protein was used.

Fig. Epithelial repair of human ex vivo wounds progresses via an extending shield mechanism. Human ex vivo wounds were cultured in the presence of the fluorescent dyes. CMFDA and PI cotreatment at time of injury (D0) identifies live (CMFDA; green) and dead (PI; red) cells around the injury site (A), and these wounds traced to day 1 indicate PI‐labeled cells are no longer present (B).

文獻(xiàn)2）Frazer J. Bye et al. Development of bilayer and trilayer nanofibrous/microfibrous scaffolds for regenerative medicine.Biomater. Sci., 2013, 1, 942-951.

Method (Cell migration into scaffolds at 7 days)：CellTracker red (CMTPX) or green (CMFDA) were applied to the hESMPs and fibroblasts respectively, prior to seeding. Cells were washed 3 times with 5 ml PBS then 10 ml of serum free cell-appropriate medium containing CellTracker (10 mM) was added and the cells incubated for 45 minutes at 37 °C. After incubation, the cells were washed 3 times with 5 ml PBS following which they were seeded onto scaffolds.Scaffolds could then be imaged in an Axon ImageExpress microscope (Molecular Devices, Sunnyvale, USA) at 570 nm λex–620 nm λem (CellTracker red) and 480 nm λex–533 nm λem (CellTracker green).

Fig. Co-culture of CellTracker™ labelled fibroblasts (green) and hESMPs (red) on bilayer membranes of either PHBV–PLA or PHBV–PCL. hESMPs were seeded on day 1 (red) onto the PHBV face of each bilayer. Fibroblasts were seeded on either the PLA or PCL face of the bilayer on day 2 (green) and then cultured for a further 7 days. In A fibroblasts (green) are confined to the PLA face after 7 days with no sign of hESMPs (red). On the opposite face (B, PHBV), hESMPs (red) are also present once again with no fibroblasts.

使用方法

1．細(xì)胞準(zhǔn)備

在合適的培養(yǎng)基內(nèi)培養(yǎng)細(xì)胞。貼壁細(xì)胞可以在含蓋玻片的培養(yǎng)皿內(nèi)爬片生長(zhǎng)，裝入足量的生長(zhǎng)培養(yǎng)基。

2．操作步驟

以下描述的是將染料加到培養(yǎng)細(xì)胞以及在熒光顯微鏡下成像的步驟。各種因素，比如將染料加載到細(xì)胞或組織，可能都需根據(jù)特定的細(xì)胞類型對(duì)某些條件做出修改。

探針的最佳染色濃度需根據(jù)用途來調(diào)整。建議剛開展實(shí)驗(yàn)需要測(cè)定至少1個(gè)10倍范圍內(nèi)的濃度。一般來說，長(zhǎng)期染色（≥3天）或使用快速分裂的細(xì)胞需5-25µM的染料。對(duì)于短期實(shí)驗(yàn)（比如活力測(cè)定），使用低濃度染料（0.5-5µM）。為了維持正常的細(xì)胞形態(tài)和降低潛在的偽影，盡可能使用低濃度的染料。

2.1制備Cell-Tracker染色工作液

①開瓶前將產(chǎn)品從冰箱取出，放到室溫使其回溫至少20min。

②用高質(zhì)量的無水DMSO溶解粉末使其濃度為10mM。例如，對(duì)1mgCell-Tracker Green CMFDA（Mw:464.86g/mol）凍干粉，加入215µlDMSO充分溶解，即得到10mM母液。

③ 用無血清培養(yǎng)基稀釋母液到0.5-25µM的工作濃度。預(yù)熱染色工作液到37℃。

2.2懸浮細(xì)胞染色步驟

① 離心收集細(xì)胞，吸掉上清液。用預(yù)熱的Cell-Tracker染色工作液輕輕的重懸細(xì)胞。

② 在適合特定細(xì)胞類型的生長(zhǎng)條件下孵育15-45min。

③ 離心細(xì)胞，吸掉Cell-Tracker染色工作液。

④ 加入選擇的培養(yǎng)基，將標(biāo)記好的細(xì)胞分配到載玻片或到選擇的培養(yǎng)器皿內(nèi)。

⑤根據(jù)附表1選擇合適激發(fā)和發(fā)射波長(zhǎng)的濾片來進(jìn)行成像檢測(cè)。

2.3貼壁細(xì)胞染色步驟

① 吸走培養(yǎng)基。

② 輕輕加入預(yù)熱的Cell-Tracker染色工作液。

③ 在適合特定細(xì)胞類型的生長(zhǎng)條件下孵育15-45min。

④吸掉Cell-Tracker染色工作液。

⑤ 加入選擇的培養(yǎng)基。

⑥根據(jù)表1選擇合適激發(fā)和發(fā)射波長(zhǎng)的濾片來進(jìn)行成像檢測(cè)。

 3．熒光顯微鏡觀察

Cell-Tracker熒光探針可用帶標(biāo)準(zhǔn)光學(xué)和圖像增強(qiáng)的各種落射熒光光學(xué)顯微鏡檢測(cè)。根據(jù)染料選擇合適的濾片。見附表1 Cell-Tracker熒光探針的光譜特征。

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— —Written/Edited by V. Shallan【版權(quán)歸MKBio懋康所有】

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