The video outlines a laboratory protocol for preparing and sterilizing sapphire discs for cell culture. It details processes including sonication, flaming, labeling, UV sterilization, and seeding HeLa cell lines on the discs until 80-90% confluency is achieved, ensuring a sterile environment for cell growth.

﻿preparing sapphire discs for cell culture

Protein Lokalizasyon Analizi için Altın Nanopartiküllerin Doğrudan Sentezi

In the postgenomic era, the development of green fluorescent protein (GFP) as a single-molecule reporter for light microscopy has revolutionized the field of modern life science research1,2. For decades, electron microscopy (EM) has been a powerful tool for intuitively observing the cellular ultrastructure with nanoscale resolution3; however, the precise identification and localization of protein molecules remain challenging.
The most commonly used EM labeling technique is the immunoelectron microscopy (IEM) labeling technique, which is based on the antigen-antibody reaction. However, although many techniques have been developed in the field of IEM labeling, including pre-embedding IEM and post-embedding IEM (on resin sections or hydrated cryosections), it still suffers from low labeling efficiency (&#60;10%)4,5, which is related to the sample preparation and antibody quality. To overcome these limitations, developing genetically encoded tags has great application potential.
Two main types of EM tags have been thoroughly explored in recent years. One type is the DAB staining method, which utilizes tags such as APEX2 to oxidize 3,3&#39;-diaminobenzidine (DAB) to osmiophilic polymers for EM visualization6,7,8,9,10,11,12. It enables the labeling of high-abundance proteins in subcellular regions but is not suitable for single-molecule counting. The other type utilizes metal-binding proteins, such as ferritin13 and metallothionein (MT)14,15,16,17,18,19,20,21,22,23,24,25,26, to generate electron-dense metal deposits in situ for EM visualization. Only the latter has real potential for single-molecule visualization and counting. The molecular size of ferritin is too large (~450 kD) for it to be used as a promising tag, whereas the small size (~5 kD) of MT and its ability to bind various ions through its 20 cysteines have attracted great attention. Several labs have tried to label purified MT-fusion proteins or MT-expressing cells by incubating directly with Au+. These attempts have initially proved that MT tags can bind gold ions to form high-contrast signals, but none have really achieved the effective identification of individual proteins in cells, and they are not widely applicable14,15,16,17,18,19,20,21,22,23.
The ANSM-based AuNP synthesis technique, which involves synthesizing 2-6 nm-sized AuNPs directly on cysteine-rich tags (e.g., MT, the MT variants MTn and MT&#945;, AFP) as electron-dense labels for EM visualization, is the first reliable and applicable approach for protein labeling and single molecule detection in cells24,25,26. It allows for the specific synthesis of AuNPs on isolated tag fusion proteins and has achieved an unprecedented labeling efficiency in nonfixed or chemically fixed prokaryotic (E. coli) and eukaryotic (S. pombe) cells. However, implementing the same protocol in more advanced systems such as mammalian cells or even tissues involves additional challenges, such as the more complex intracellular redox homeostasis and more fragile cellular structure.
This study presents a clonable EM labeling technology, which combines the novel ANSM-based AuNP synthesis technique for labeling genetically encoded cysteine-rich tags (MT) with the HPF/FSF-rehydration-HPF/FSF sample preparation method, enables the unambiguous single-molecule identification of tagged proteins in the ER membrane, ER lumen, and mitochondrial matrix in HeLa cells. The current method combines the characteristics of high labeling efficiency, a high signal-to-noise ratio, single-molecule labeling, and strong universality, and this method has broad application prospects in life science research.

Yazar Spotlight: İyi Korunmuş Ultrastructure ile Protein Lokalizasyon Analizi için Hücrelerde EM-Görünür Altın Nanopartiküllerin Doğrudan Sentezi  

İyi Korunmuş Ultrayapı ile Protein Lokalizasyon Analizi için Hücrelerde EM-Görünür Altın Nanopartiküllerinin Doğrudan Sentezi

Watch this Scientific Journal Video about ï»¿Preparing Sapphire Discs for Cell Culture at JoVE.com

Preparing Sapphire Discs for Cell Culture  

Safir Disklerin Hücre Kültürü için Hazırlanması

Üç milimetre x 0.16 milimetre safir diskleri, bir mililitre etanol içeren iki mililitrelik bir santrifüj tüpüne aktarın ve 10 dakika boyunca ultrasonik bir temizleyicide sonikleştirin. Her safir diski, yüzeyde görünür birikintiler kalmayana kadar bir alkol lambası alevinde yakın. Solvente dayanıklı bir kalem kullanarak her safir diskin bir tarafını bir sayı ile etiketleyin.Etiketli safir diskleri, etiketli tarafı yukarı bakacak şekilde 35 milimetrelik bir kültür kabının altına yerleştirin. Hücre kültürü kabını ultraviyole ışık altında safir disklerle 30 dakika sterilize edin. Safir diskleri cımbızla çevirin, etiketli tarafın aşağı baktığından emin olun ve ultraviyole ışık altında 30 dakika daha sterilize edin.Hazırlanan safir disklerde MTn etiketlerini ifade eden kararlı HeLa hücre hatlarını tohumlayın ve bir hücre inkübatöründe% 80 ila% 90 akıcılığa kadar büyütün.

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JoVE Journal

Biology

﻿Preparing Sapphire Discs for Cell Culture

197 Görüntüleme. Üç milimetre x 0.16 milimetre safir diskleri, bir mililitre etanol içeren iki mililitrelik bir santrifüj tüpüne aktarın ve 10 dakika boyunca ultrasonik bir temizleyicide sonikleştirin. Her safir diski, yüzeyde görünür birikintiler kalmayana kadar bir alkol lambası alevinde yakın. Solvente dayanıklı bir kalem kullanarak her safir diskin bir tarafını bir sayı ile etiketleyin.Etiketli safir diskleri, etiketli tarafı yukarı bakacak şekilde 35 milimetrelik bir kültür...

Video: Preparing Sapphire Discs for Cell Culture  

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.