对于样品制备与TEM分析低细胞数量的另一种方法
Summary
Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.
Abstract
透射电子显微镜（TEM）提供了细胞组织和超微结构的细节。然而，稀有细胞群，尤其是细胞悬浮液，如造血干细胞（HSC）的TEM分析，仍然很有限，由于高细胞数的样品制备过程的要求。有用于从稀少样品TEM分析几个细胞离心涂片或单层的方法，但这些方法无法从细胞的数量有限得到显著定量数据。这里，对于在TEM研究的样品制备的替代的和新颖的方法是罕见的细胞群，使定量分析说明。
相对较低的细胞数量， 即10000造血干细胞，成功地应用于相比，数以百万计通常用于TEM研究细胞的TEM分析。特别是，多聚甲醛，戊二醛后进行伊文思蓝染色（PFA-GA）固定形象化狭小的牢房像素让，这有利于在琼脂糖嵌入。在超薄切片观察到大量细胞群。细胞有保存完好形貌，并高尔基复合体和几个线粒体的超结构细节也清晰可见。这种高效，容易和可重复的协议允许从低细胞数的样品制备和可用于对从有限的生物样品稀有细胞群的定性和定量TEM分析。
Introduction
细胞的超微结构和亚细胞器的细节已通过透射电子显微镜（TEM）从组织或细胞沉淀1,2-研究中主要显露。组织坚固件可以很容易地用于电子显微镜研究。然而，TEM分析1,3悬浮细胞仍然充满挑战和必要的高细胞数， 即数百万个细胞。正因为如此，在悬浮稀有细胞群的超结构分析， 例如，造血干细胞（HSC），不容易评估。对于使用细胞离心涂片或单层方法稀少样品TEM分析多次尝试未能从细胞的数量有限得到显著定量数据。因此，高泡孔数的要求限制了使用这一强大的工具的理解稀有细胞群的亚细胞超微结构的信息。
为TEM研究的一个重要的限制与细胞数目有限S IN悬挂是细胞处理的定位和由此，从有限的细胞，特别是小尺寸的TEM研究仍然充满挑战。几种替代方法已被采用，以对付这种限制：BSA /双丙烯酰胺（BSA-BA）细胞悬浮液的介导聚合，将细胞与染料以使它们在薄膜支持，包括盖玻片可见的染色， 透射电镜和从细胞离心制剂4-6分析。然而，非常有限的成功达到了，因为很少有细胞在超切片后各部分找到。识别稀疏细胞的主要挑战仍然存在，因为单层细胞保持在固化的凝胶切片大多不可见的。此外，细胞的细胞离心涂片器制剂可以改变它们的细胞组织由于细胞扩散和细胞结构的脆弱性。因此，这些固有的缺陷认股权证以罕见的细胞群与更多的执行TEM研究的新方法一致性。为了克服这个问题，我们已经描述了一种新的和替代的样品制备方法用于从稀有细胞群7 TEM研究。
这里，我们报告的有效样品制备协议从稀少生物样品具有一致的定性和定量的结果执行的TEM。固定后进行伊文思蓝染色来本地化来自低数量的细胞， 即 10000骨髓造血干细胞和祖细胞，否则仍然可见一个微小的细胞沉淀，并将沉淀物嵌入琼脂糖之前脱水和树脂包埋过程。此方法聚类细胞一起，使超微结构和造血干细胞的亚细胞组织（标识为林–的Sca-1 +的c-Kit +的Flt3 – CD34 – ; HSC）的有效分析，难得0.2 – 0.5％的细胞群在骨髓中。这一实验亲母育可以进行超结构的研究，并取得许多罕见但非常重要的群体的定量结果非常有用。
Protocol
Representative Results
Discussion
该方法能够在低细胞数目TEM分析，通过使用伊文思蓝染色和琼脂糖嵌入脱水，树脂包埋期间本地化一个微小的细胞沉淀和切片工序。重要的是，它保持细胞簇一起，并保留在细胞的超结构，这是可取的，以检查数据的量化的多个小区。
在TEM研究，含有数百万个细胞的细胞团，常常需要用于有效埋入到琼脂糖/明胶基质和用于洗涤，脱水和渗透到从大量细胞中获得的数据。研究?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank the Pathology Research Core for assistance with electron microscope analysis studies at Cincinnati Children’s Hospital Medical Center. The work was supported by NIH (American Society of Hematology Bridge award to-MDF; R01 DK102890 to MDF).
Materials
| Paraformaldehyde 20% Aqueous Soln | Electron microscopy Sciences | 15713 | CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood. |
| Gluteraldehyde 70% Aqueous Soln | Electron microscopy Sciences | 16350 | CAUTION Toxic via inhalation, skin contact. Wear gloves, use fume hood. |
| Cacodyladate buffer | Electron microscopy Sciences | 12300 | Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4°C. CAUTION Irritant by inhalation or skin contact, wear gloves and work in fume hood. |
| Evans Blue | Sigma | E-2129 | |
| Low melting agarose | Sigma Aldrich | A-5030 | |
| Osmium tetraoxide | Electron microscopy Sciences | 19130 | 1 gm in 50 ml of 0.1M Cacodyladate buffer to make 2% OsO4 stock, CAUTION Very toxic by inhalation or skin contact, strong oxidizer, wear protective clothing, eye protection, use fume hood. |
| LX-112 Embedding Kits (LADD®) | |||
| DDSA (Dodecenylsuccinic anhydride) | LADD | 21340 | Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30 |
| NMA (Nadic methyl anhydride) | LADD | 21350 | |
| Epoxy Resins Epon 812 (LX-112) | LADD | 21310 | CAUTION Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood. |
| DMP-30 (2,4,6 -Tri(dimethylaminomethyl)phenol) | LADD | 21370 | |
| Reynolds lead citrate (EM Stain II) | Leica | CAUTION Toxic by inhalation, avoid contact with skin or eyes. | |
| Uranyl acetate (EM Stain I) | Leica | 8072820 | CAUTION Toxic by inhalation, avoid contact with skin or eyes. |
| Iscove's Modified Dulbecco's Medium (IMDM) | ATCC | 30-2005 | composition: NaCl, NaHCO3, HEPES, Glucose, Ca, Mg, sod pyruvate etc. For for details https://www.atcc.org/~/media/Attachments/E/9/7/E/4893.ashx |
| Pyramid tip mold | Ted Pella | 10585 | |
| mounting cylinders | Ted Pella | 10580 | |
| Ultra-microtome | (Leica EM UC7) | ||
| 200 mesh grids (nikil) | Electron microscopy Sciences | G-200 Ni | |
| Electron Microscope | Hitachi model H-7650 | ||
| Image capture engine software | AMT-600 | ||
| 35 mm plastic petri dishes | Fisher scientific | ||
| Quick Bond Super glue Cyanoacrylate | Electron microscopy Sciences | 72588 | |
| Razor blades | |||
| Glass scintillation vials | Fisher scientific | 03-337-14 | |
| Glass slides | |||
| Eyelash tool | |||
| Metal Loop tool |
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