Flow Board Chapter 3
Chapter 3 Gourmet Food Heir (Arc 1.2) Jing Yang opened the system’s numerical adjustment functions, in addition to things like appearance, temperament, and physical condition, a countless number of various skills could also be adjusted. He was too lazy to go through and choose the items one by one, instead simply letting the system automatically adjust for him according to the desires of the original owner. When he heard the doorbell ring, Jing Yang got up from bed and opened the door. Chen Rui looked at Jing Yang, who had disheveled hair and was still wearing a nightgown, and asked doubtfully, “You just got up? Didn’t you really pay attention to this auction? Earlier you even did a lot of preparation just for today, now that it’s finally here, you’re actually no longer concerned about it anymore?” Jing Yang pushed at his disheveled hair and said to Chen Rui, “You sit for a bit, I’m going to go wash up.” While Jing Yang washed up, he brought up the information related to Chen Rui. Chen Rui was Lu Xuexian’s maternal aunt’s grandson, which meant he’s also Lu Jingyu’s distant cousin only younger than him by a few months.
But his parents died early, and he was brought up by Lu Jingyu’s mother’s aunt. Since childhood, Chen Rui had a good relationship with Lu Jingyu, they were classmates and friends, as well as cousins.
When Lu Jingyu was framed and chased because of offending Zheng Junming, only he (CR) still accompanied and supported him (LJY), but his ending was also not great In a man-made car accident, Chen Rui was killed while trying to protect Lu Jingyu. At that time Lu Jingyu held the blood-soaked Chen Rui waiting for the ambulance to come, but even after a long time it still didn’t show up. With his last breath, Chen Rui told Lu Jingyu that he had to keep living well, that he should live even better than that Zheng Junming.
At that time Lu Jingyu was endlessly grieved, he hated that his ability wasn’t enough and even needed Chen Rui to sacrifice his own life to protect him, but he didn’t even have enough ability to help Chen Rui revenge From now on, he, Jing Yang, is Lu Jingyu, and Lu Jingyu’s every desire, he would take them as his own business to complete. After he finished washing up, Jing Yang opened the fridge to prepare breakfast, luckily even though the original Lu Jingyu wasn’t that great at cooking, but he still had the habit of cooking, so the fridge was pretty well stocked. “You’re still going to make breakfast?” Chen Rui was surprised and asked, “The auction is going to start very soon, if we don’t leave now we’re going to be late.
Are you not planning to go?” “We won’t be late, and even if we did get there late, it’s no big deal.” Jing Yang took out some meat and eggs, scallions, vegetables, and other ingredients. There were still some leftovers in the rice cooker, so he prepared to make some fried rice.
He’s been a chef for several lifetimes, even though his cooking is pretty good, it couldn’t be considered amazing. Now he used the system to adjust his cooking skill to the highest, and wanted to test out the effect. Chen Rui saw him calmly actually starting to make breakfast, and was still very doubtful. He dragged over a high stool to sit across from Lu Jingyu at the table where he was cutting vegetables. “Bro, I found that you seem to be a little different today.” Putting the chopped vegetables to one side, Lu Jingyu was preparing to start cutting the meat when he raised his head to look at him (CR). “Where am I different?” “I thought that you drank too much last night and now should have been in a hungover state, at least with a dispirited expression, but for some reason you actually look radiant, as if you’ve become even more handsome than before.” Chen Rui wasn’t really trying to compliment him, but truly felt that he looked different, carefully observing his state, it seemed like he had changed into a different person.
“Thanks for the compliment, but I didn’t become more handsome, rather I have always been this handsome.” Jing Yang turned to face the stove and placed the ingredients into the pot. Lu Jingyu inherited his mother’s appearance, so naturally don’t need to mention his features being exquisite and handsome, but because he started drinking since he was in his teens, his body had long been ruined by all the alcohol, and then adding on his stifling dejection, his entire being seemed to be shrouded in depression and gloom. Obviously he was only a youth in his early twenties, yet his face was covered in a layer of vicissitudes. Now, however, the system had not only raised his cooking ability, but also improved the health of his body, along with even his charisma and temperament. So for Chen Rui, who was very familiar with Lu Jingyu, he would feel like he had become a different person.
“Smells so good.” Chen Rui sniffed. “What kind of fried rice did you make, why would it be so fragrant?” “Just some ordinary fried rice.” Jing Yang scooped the fried rice in the pot into two plates, passing one to Chen Rui. “Try it and see how it tastes.” While he was making the fried rice, Jing Yang already felt an obvious promotion in his cooking, whether it was his chopping skills for the meat, or his control of the fire temperature and timing. There were even other techniques he had never known about that his brain would subconsciously control his body to complete. Chen Rui looked at the fried rice with its distinctly separated grains, the mix of vegetables and meat stimulated his appetite. When he tasted a mouthful and had only chewed a few times, he closed his eyes with emotion. “It’s too good!
Bro, this is definitely the best fried rice I’ve ever eaten in my life.” Jing Yang tried a couple mouthfuls and also felt totally satisfied. The rice grains looked fresh, not greasy at all, and after chewing, basically every grain of rice brought the taste of the meat juices and vegetable flavor. Jing Yang had been a chef before, so he knew that the simpler the dish, the harder it is to make it especially delicious. For this fried rice, he could give himself a 10 rating without any modesty. As he thought about some dishes that he had wanted to do previously but never knew how to make well, the processes and details of the recipes instantly appeared in his mind.
He smiled, this system was truly useful, even the dishes that were already lost in this world could be found. Chen Rui scraped his plate totally clean, without leaving behind even a half grain of rice, and even instantly changed his habit of drinking water immediately after eating, since he couldn’t bear washing away the flavor remaining in his mouth. “Bro, were you possessed by the kitchen god today?
I’ve eaten your food so many times, your skill today was really really really really amazing.” “You stuttering?” Jing Yang looked at him laughingly, conveniently cleaning up the kitchen. “I’m just using a forceful tone to express how delicious your fried rice was.” “Let’s go, weren’t we going to the auction?” Jing Yang wiped his hands clean and took his coat to leave. Because the fried rice was too good, Chen Rui had already forgotten all about the auction. Now he just remembered about it and hurriedly followed him out the door.
The auction that Chen Rui had kept on mentioning was a property auction, and was also a trap especially set up for Lu Jingyu. Before Lu Xuexian committed suicide, she found a lawyer to transfer all the properties that she controlled to Lu Jingyu’s name, and once Lu Jingyu came of age, he would be able to inherit them immediately, so he was currently still quite rich. It was not until he jumped into the trap that others had prepared for him that his eternal damnation started. As long as something was related to Zheng Junming, Lu Jingyu would always be obsessed with it.
He heard that Zheng Junming was interested in a certain piece of land, planning on constructing the city’s largest restaurant on it and calling it Zheng Ji Restaurant. Once he thought of that father and son using the Lu family’s cooking skills yet using the name of the Zheng family, his heart felt very uncomfortable. Under the deliberate instigation of a few of his drinking buddies, Lu Jingyu decided to take down this piece of land, building and operating a restaurant there himself. In order to take down this piece of land, he paid a very high price, spending almost all of his funds. In order to build the restaurant, he had even sold the shares and several properties that his mother had left for him. It was not until his restaurant had been half built that it was suddenly exposed that this piece of land had been a mass grave a long time ago, and underneath the ground was a pile of skeletons. The restaurant construction was forced to shut down, because even if the construction was finished, there would be no one willing to eat on top of a pile of dead bones.
So Lu Jingyu suffered a tremendous loss, this matter was obviously a trap someone deliberately caused for him, otherwise why would the news suddenly explode only when the restaurant was almost completed. When constructing the building, they definitely have to excavate the foundation, when excavating how would they not be able to find the skeletons.
Yet this was actually concealed from Lu Jingyu, from the contractors down to the workers, not a single person told him about this matter. This was obviously a scam especially prepared for him. When the two got to the auction, one of Lu Jingyu’s drinking buddies Xu Peng was standing outside, wearing an anxious expression waiting for them. Once they got off the car, he immediately greeted them. “You guys finally came, the auction has already started, let’s quickly go in.” A lot of people were already sitting in the auction hall, a few of them were visibly relieved when they saw Lu Jingyu come in the door. These people were sent by the Zheng family father and son, when they saw Lu Jingyu kept on not showing up, they were truly concerned that he suddenly wouldn’t come today. When other pieces of land were being auctioned, Lu Jingyu seemed to be uninterested, boredly flipping through the brochure.
It was not until that piece of land started being auctioned that he sat upright, put on a solemn expression, and showed an appearance of being determined to win the land. The several people that the Zheng family father and son had dispatched exchanged glances with each other and showed ridiculing smiles, at that moment the Lu Jingyu in their minds was just a stupid and gullible idiot. Their task this time was totally simple, they just needed to casually sit there and raise the price, waiting for him to be fooled. At the beginning a lot of people were calling out prices, but the price was constantly being raised, and had already gone past its original value, so the number of people calling had slowly decreased. In the end, only Lu Jingyu and the Zheng family pair’s people were still constantly bidding. Because those people sat pretty far away from Lu Jingyu, and there were a bunch of people between them, they didn’t need to hide their expression, obviously revealing the joy of almost being able to succeed.
They shouted out one last high price and waited for Lu Jingyu to increase the bid. Xu Peng sitting next to Chen Rui was equally as excited as those several people, if Lu Jingyu succeeds in taking down that piece of land this time, he would be able to receive a very large sum.
His eyes seemingly seeing this money immediately getting to his hands, his heart was extremely excited, but because he was too close to Lu Jingyu, in order to not be revealed, he could only control his excitement. These people harboring evil intentions were all excitedly waiting, yet kept on not hearing Lu Jingyu’s bid. They thought this was somewhat strange and looked over there doubtfully. Chen Rui also looked at Lu Jingyu, using his eyes to ask him whether he was going to continue to bid. Lu Jingyu shook his head, and Chen Rui stopped yelling out a bid. “Jingyu, call out a bid, that piece of land is quickly going to be taken away.” Xu Peng and Lu Jingyu were separated by Chen Rui in the middle, so he could only lean forward to whisper to Lu Jingyu.
Lu Jingyu glanced over then didn’t pay any attention to him. The auctioneer on stage continually asked if there were any more bids, but Lu Jingyu and Chen Rui never made any sound. On the way there, Lu Jingyu had already told Chen Rui his limit on the price, and had told him that once this price was exceeded, they definitely couldn’t continue bidding, otherwise they would be doomed. Chen Rui also felt that this price was much too high, if it became even higher, they would rather not have this land. “Jingyu?” Xu Peng anxiously looked at the auctioneer on stage, then leaned forward again to look at Lu Jingyu, worriedly saying “Quickly bid, otherwise it will be taken by someone else.” “It’s not you who wants to buy the land, what are you so worried about?” Chen Rui pushed him away to make him sit upright, he had long disapproved of Xu Peng, these drinking buddies spent all their time thinking of ways to take advantage of Lu Jingyu’s money. Those several people kept on not hearing Lu Jingyu bid and were already starting to worry, they couldn’t help frequently glancing over there, their hearts were like cats on a hot roof, more and more impatient and uneasy. Lu Jingyu had already decided his highest price before, Xu Peng had leaked this out earlier, but right now the price was not the same as the one that he had leaked.
It’s not a big deal if he doesn’t get that money, but if he offends someone he can’t afford to offend that would be a big matter. If Lu Jingyu doesn’t take down this piece of land, he would definitely not have a good end. In a moment of desperation, Xu Peng’s brain heated up and he prepared to raise his hand to bid on behalf of Lu Jingyu. Translator Notes: 1 calm – 不慌不忙, idiom, literally calm and unhurried 2 look radiant – 容光焕发, idiom, literally face shining/glowing 3 without any modesty – 毫不谦虚的, literally what it means, I don’t think it’s in a bad way, but the English does seem to have an unfavorable connotation lol 4 determined to win – 势在必得, idiom, literally force to certainly obtain 5 harboring evil intentions – 心怀叵测, idiom? MDBG brought me right to 居心叵测, an idiom which has the same meaning 6 cat on a hot roof – 热锅上的蚂蚁, idiom, means anxious/agitated 7 in a moment of desperation – 情急之下, idiom, literally an inspiration during desperation Update: Mandatory celebratory post for finally finishing my massive exam! (ﾉ◕ヮ◕)ﾉ.:･ﾟ✧ Honestly, an 8 hour test really takes a toll on you. At least it’s all over.
I’ll probably be updating every Sunday and Thursdayish in the morning, though the schedule might change for random reasons. Schoolwork can be a liability sometimes. Random Notes: I actually kept track of the time I took translating this chapter. I thought it’ll be interesting to see how much time I’ve been spending on each chapter.
For this one, it was from 3.6k characters to 2.5k words, 1:30 pm – 3:30 pm. Of course, this doesn’t include me formatting later and then rereading for typos and very slight edits. 2 hours isn’t too bad.
Luckily for the rest of the arc there’s only one other chapter that has 4k characters, and a couple that have 3.8k, so most of them should be around 1.5-2 hours, at least for the first arc. Something I’m pretty annoyed by is that I quite like this theme but the body text is just so small. But bc free WP is free, they only have 5 different text sizes, and the next one is suddenly huge.
I’ve been using MarsEdit to write out all the text, and then format back on WordPress. I’ve only recently noticed for this chapter that when I come back to WordPress to do the final edits on the text saved from MarsEdit, the text size seems to stay from MarsEdit. Does this mean that I can change the text size? All the possibilities!
I’ll have to test this out next time. Also, while I’ve been translating, it’s become strangely satisfying connecting sentences with commas. Hopefully this doesn’t ruin my English writing later (´･ᴗ･ ` ).
3.1 Introduction When a compound absorbs light (and hence energy) electrons are raised from the ground state to an excited state. Electrons return to the ground state by a variety of transitions which may involve the emission of a quantum of light (radiative transition). This effect is termed fluorescence ( Figure 3.1). The emitted light will always be of lower energy, and hence longer wavelength, than the exciting light. The energy may also be lost by nonradiative processes and eventually dissipated as heat. Figure 3.1. The absorption and emission of light during fluorescence. After excitation by light absorption, the electron moves to an excited state of lower energy (red arrow).
When it drops to the ground state (green arrow) light is emitted. Because the colour of the exciting and emitting light are different, they can be separated from one another by using optical filters.
Fluorescence detection is a sensitive technique because a positive signal is observed against a negative background. The detection of up to 14 compounds fluorescing at different wavelengths permits multiparametric analysis of cells and has greatly increased the power of flow cytometry.
The majority of applications use up to four fluorescences. 3.2 Properties of a fluorochrome The important properties of a fluorochrome are its absorption spectrum, its extinction coefficient at a wavelength convenient for excitation, its emission spectrum and its quantum efficiency. The latter is the number of photons emitted for every photon absorbed. The difference between the maxima in the wavelengths of absorption and emission is known as the Stoke’s shift. The higher the Stoke’s shift, the greater the separation between the exciting and the emitted light. The properties of a fluorochrome will depend on its environment.
For example, propidium iodide (PI), which is used to stain DNA, is only weakly fluorescent in water; on intercalating with DNA, the fluorescence increases 50 fold due to the hydrophobic environment. Some fluorochromes, such as fluorescein, are sensitive to pH. There are other compounds whose fluorescent spectrum changes according to the concentration of certain ions, such as Ca (see Section 3.3.3). Fluorescence can also be quenched by an interaction with another molecule in which the energy is dissipated by a non-radiative transition. This can be illustrated by the bis-benzimadazole dye, Hoechst 33342, which binds to DNA giving blue fluorescence on excitation with UV. However, if DNA is labelled with 5'-bromodeoxyuridine, the fluorescence of the dye is quenched by the bromine atom. If a compound is over-labelled with a fluorochrome, fluorescence can also be quenched by interactions between the molecules of fluorochrome.
This effect can be observed when liposomes are loaded with fluorescein; fluorescence decreases with increasing dye concentration above about 20 µM. If two fluorochromes are closely associated, energy transfer can occur whereby excitation of one compound causes the other to fluoresce. For example, if the rhodamine derivative, Texas Red, is bound to the fluorescent protein, phycoerythrin (PE), excitation of PE will lead to emission of light from Texas Red.
The effective emission maximum will have been shifted from that of PE (576 nm) to that of Texas Red (620 nm). For energy transfer to occur, the acceptor molecule has to have an absorption spectrum that overlaps with the emission spectrum of the donor molecule ( Figure 3.2).
The most common fluorochrome used to label proteins is fluorescein isothiocyanate, the isothiocyanate group reacts with the amino groups on the lysine residues in the protein. The number of labelled proteins that can be used has been greatly extended by the use of tandem dyes. Examples include conjugates of PE and allophycocyanin (APC) with various cyanine dyes ( Table 3.1).
A wide range of directly labelled monoclonal antibodies and labelled anti-Igs are available from several manufacturers. It is seldom necessary to label proteins in the laboratory and the reader is recommended to purchase labelled reagents. 3.3.2 Quantum dots Quantum dots are fluorescent nanocrystals; those used in flow cytometry are 10 - 20 nm in size. They have a wide absorption spectrum and so can be excited by a range of different wavelengths. Their emission wavelength depends on their size and can be from blue through to deep red. Their emission spectra are very narrow compared to the emission spectra of conventional dyes, reducing the overlap between the different fluorescences. (The significance of this is discussed in Chapter 5).
Their use is particularly attractive in immunology enabling a large number of T cells subsets to be enumerated in a single experiment (Chattopadhyay, 2006). At present, their use is limited but is certain to increase as the technology for linking them to different probes improves and the price reduces. The fluorochromes described in this section bind stoichiometrically to nucleic acids so that they can be used for quantitative measurement. This is essential for the measurement of ploidy and the cell cycle (see Chapter 6). The anthraquinone, DRAQ5, and bis-benzimadazoles, such as Hoechst 33342, cross an intact plasma membrane and can be used to visualize the cell cycle in viable cells.
With the other compounds listed, cells have to be fixed or permeabilised before staining for DNA. Propidium iodide (PI) is the dye most widely used as a DNA stain. PI (and the closely related compound, ethidium bromide) intercalates between the bases in double stranded nucleic acids.
Flow Board Chapter 3 Lesson 1
It is excited by blue light giving red fluorescence. With an argon-ion laser tuned to 488 nm, PI can be used in combination with fluorescein, which makes it particularly useful for simultaneous measurement of antibody binding and DNA content. PI also binds to double stranded RNA, which has to be removed by treatment with RNase. DRAQ5 has an absorption maximum at 646 nm. However, it has a broad absorption spectrum and a high extinction coefficient and it absorbs sufficient light at 488 nm to permit its use with an argon-ion laser. The bis-benzimadazoles (usually referred to by the number given to them by the original manufacturer, Hoechst) bind preferentially to AT-rich regions in the small groove of double-stranded DNA and fluoresce blue when excited by UV light. The antibiotic, chromomycin A3, binds to the GC-rich regions of DNA.
It has been used in conjunction with Hoechst 33258 to resolve chromosomes of similar size but with different AT/GC ratios (see Chapter 9, Section 9.12). 4',6-diamidino-2-phenylindole (DAPI) has been used extensively to label DNA for excitation with UV; it is often the stain of choice in instruments employing a mercury arc lamp. A new series of cell-permeant dyes has recently been introduced by Invitrogen with the proprietary name, Vybrant DyeCycle (VD). VD Violet can be excited either with UV or with a solid state violet laser at 410 nm.
VD Orange can be excited at 488 nm or at 530 nm. 3.3.5 Other reporter molecules Some applications using other reporter molecules are outlined in Chapter 10. A few general principles are described below. Loading the cell. The internal milieu of the cell can be explored using compounds whose fluorescence is sensitive to the environment. Such reporter molecules must be introduced into the cell and, once there, they should not migrate out. This is not a problem if the probe binds tightly to a cell component (e.g. Hoechst 33342 to DNA) or if the partitioning of the probe between the cytoplasm and the medium is being measured (e.g.
Probes of plasma membrane potential, see Chapter 9, Section 9.3.4). With other probes a different strategy must be found. Many reporter molecules are charged and, as such, have difficulty in crossing the plasma membrane; a general procedure adopted for loading cells is to mask the charge by preparing an ester. The uncharged molecule will diffuse into the cell in which non-specific esterases convert the reporter molecule to its original non-permeant form ( Figure 3.3). Method for loading charged dyes into cells. Figure 3.4. The change in emission spectrum of indo-1 with change in calcium concentration; excitation wavelength: 360 nm. Redrawn with permission from Rabinovitch and June (2000).
Flow Board Chapter 361.1
Ratio measurements. Once in the cell, the amount of a reporter molecule present, and hence its total fluorescence, will generally depend on the size of the cell. Changes in the amount of light emitted on a single cell basis cannot be measured; only a general shift in the population can be recorded. It is therefore useful to use a probe that shows a shift in the wavelength of emission. Fluorescence is measured at two wavelengths and the ratio calculated for each cell. This ratio will be independent of the total amount of fluorochrome in the cell. For example, the calcium indicator, indo-1, has an emission maximum of about 490 nm in the absence of Ca; in the presence of 1 mM Ca, this shifts down to about 410 nm ( Figure 3.4). It can be loaded into cells as its acetoxymethyl ester.
Indo-1 is excited with UV and changes in the concentration of intracellular Ca recorded by observing fluorescence either side of the isobectic point (for example, at 400 and 520 nm). Reporter molecules showing a shift in emission spectrum have also been used to measure intracellular magnesium ions and pH (see Section 10.4). References Chattopadhyay, P.K. (2006) Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Rabinovitch, P.S. And June, C.H. (2000) Intracellular ionised calcium, magnesium, membrane potential and, pH.
In: Flow Cytometry. A Practical Approach. 3rd edition, (ed. Ormerod, M.G.), pp. Oxford University Press, Oxford.
The online catalogue from Invitrogen is good source of information about most fluorescent probes used in flow cytometry. For information about DRAQ5, see the Biostatus Web site.