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Description
Organotial Human Normal esophageal organoid Culture KitProduct Specification Description Composition: Component name Specification Human esophagus cancer organoid medium A 100mL Primary culture buffer B 250mL Human esophagus cancer primary tissue digestive juice C 30mL Organoid passage digestive juice D 30mL Tissue preservation solution E 100mL Organoid cryopreservation fluid F 20mL Organoid subculture buffer G 250mL Instructions 1, primary (1) The harvested tissues should be placed in precooled (2 8 C)
Product Specification
| Description |
Composition:
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| Instructions |
1, primary (1) The harvested tissues should be placed in precooled (2-8° C) In the sampling bottle of tissue preservation solution E, the tissue was quickly transferred to a clean laboratory for tissue processing and cell separation, photographed and recorded. (2) Prepare several culture dishes and add primary culture buffer B precooled at 4 ° C for later use. (3) The sampling bottle was disinfected, and the tissue was placed in the Petri dish. After washing three times with primary culture buffer B, the tissue was cut into tissue blocks of about 1-3mm3in volume using ophthalmic scissors or scalpel. (4) The tissues were digested with normal human esophageal primary tissue digestion solution C and shaken at 37℃ for 20-25min (the digestion was observed at any time during the digestion). (5) A small amount of liquid was observed under the microscope. When more single cells or cell clusters below 70um were observed under the microscope, triploid volume primary culture buffer B was added to terminate the digestion. (6) The filtrate was filtered by a sieve with a pore size of 100um, and the filtrate was collected. After enrichment and centrifugation at 300g for 5min, the supernatant was removed, and the primary culture buffer B was added and resuspended for centrifugation. (7) Matrix glue calculation: The volume of collected tissue was observed after step 6, and matrix glue (abs9495) was added 25 times the tissue volume to resuspend the paving plate. (8) For the example of 24-well cell culture plates, 25uL of tissue matrix glue mixture was dispensed to each well for paving (operation at 4 ° C). (9) The paved culture plate was placed in an incubator at 37 ° C for 10-15min to become gelation, and human normal esophageal organoid medium A was added for culture (return to room temperature). 2, Organoid subculture (1) The medium was aspirated by pipetting gun, and 1-2mL of 4 ° C organoid subculture buffer G was added to each well and placed for 2min. (2) The matrix glue was gently blown by a pipetting gun, collected in a 15mL centrifuge tube and allowed to rest for 10min at 4 ° C. (every 6-8 Wells were a group) (3) a: When the number of organoids was insufficient or the volume was small: the supernatant was discarded by centrifugation for 5min, then an appropriate amount of organoid passage culture buffer G was added and resuspended into a 1.5mL centrifuge tube, and the liquid was discarded by centrifugation at 300g for 5min for the fourth step. b: When organoids are large in number or size: The supernatant was discarded by centrifugation for 5min, then the organoid passage digestion solution D was added for 2-3min, then the organoid passage culture buffer G was added to terminate the digestion, the mixture was discarded by centrifugation for 5min, then the organoid passage culture buffer G was added and the mixture was resuspended and transferred to a 1.5mL centrifuge tube, and the liquid was discarded by centrifugation at 300g for 5min for step 4. (4) After the organoid was collected, 25uL of Matrigel was added to the 24-well cell culture plate, and 500uL of human normal esophageal organoid medium A was added to the incubator for 10-15min. 3. Organoids were frozen (1) The medium was removed by suction with a pipetting gun, and 1-2 ml of 4℃ organoid passage culture buffer G was added to each well and placed for 2min. (2) The matrix glue was gently blown by a pipetting gun, collected in a 15mL centrifuge tube and allowed to rest for 10min at 4 ° C. (every 6-8 Wells were a group) (3) The supernatant was discarded by centrifugation for 5min, then an appropriate amount of organoid passage culture buffer G was added and resuspended again, and the liquid was discarded by centrifugation at 300g for 5min. (4) Add appropriate amount of organoid cryopreservation solution F, gently blow and resuspend, take 24-well cell culture plate as an example: the density is 2 Wells and freeze 1 tube, each tube volume is 1.4mL. (5) After making the labeling information and cooling, the samples were transferred to liquid nitrogen for long-term storage. 4, organoid resuscitation (1) 10mL organoid subculture buffer G was placed in a 15mL centrifuge tube. (2) The frozen organoid cells were removed from the liquid nitrogen tank and quickly thawed in a 37℃ water bath. (3) During the water bath melting process, it is necessary to gently shake the freezing tube to ensure that the frozen solution is completely melted within 1-2min. (4) The dissolved organoid cells were quickly transferred to a 15mL centrifuge tube, gently blown with a pipette for 6-8 times, centrifuged at 300g for 5min, and then the supernatant was removed and the organoid cell precipitate was collected. An appropriate amount of organoid passage culture buffer G was added and resuspended into a 1.5mL centrifuge tube and centrifuged at 300g for 5min. (5) Matrigel was resuspended, 25uL of Matrigel per well was spread in A 24-well cell culture plate, placed in an incubator for 10-15min to gel, and 500uL of human normal esophageal organoid medium A was added. |
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| Storage Temp. | Stored at 4 ° C, valid for 3 months; Stored at -20 ° C, valid for 1 year, see label for details. |
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4.4 ★★★★★
Based on 24 reviews
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Product Reviews
★★★★★ 4
Interesting
Format: Kindle
This was a very captivating book once you got into it thoroughly. But the third person perspective was a bit hard to get used to. But as you got into it and followed the different characters, it was interesting and filled with intrigue, conflict and forbidden love.
I can’t wait to read the next one and to complete the series.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 12, 2022
★★★★★ 5
OUTSTANDING
Format: Kindle
This was very, very good. The world is vast and characters are complex. There is a good plot with a whole lot going on. This is well written. Good twists and turns and some heart breaking moments. You will love these characters, they have heart and loyalty. I am hoping that there will be several more books. We've yet to see anything from the Sea Court but only a mention of them here and there. The Wood Court was given a quick couple of scenes, and only as far as some warriors, we've yet to enter their court and the Shadow Court, I'm not sure if they will be a force for good or bad, but they definitely will play a much bigger role moving forward. This is primarily the Ice and Air Courts. Told in multiple views, which I loved, it gives you a chance to see things from different eyes. There's alot of political maneuvering and deception. I loved it and will pick up the next book as it becomes available. If you like The Fae and the courts, you should love this. I think the author has mucn in store for us.
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Reviewed in the United States on March 8, 2020
★★★★★ 4
Definitely worth the read!
Format: Kindle
After taking a deep breath and taking in that wicked twist of an ending, I have finally composed myself. My first thought when I started this book was that I love Reyna's character. I was intrigued by her connection with her familiar and the Ruin that is plaguing her land. It came as no surprise that she took her sister's place in an attempt to protect her. When they reach the Air Court everything slows down. This is where it was iffy for me. First of all, I like multiple POV's in books however 7 is a bit much. It starts to interrupt the story line. I felt like I was finally making progress connecting with one character, then it was switched to another person. I felt they all had necessary or pertinent information but not necessarily were they all POV worthy. The only other thing that annoyed me was that Reyna constantly was " trapped." She would rush off without thinking, only to need rescuing. She is brilliant in a fight, but she really doesn't think through anything. Lorcan is amazing. I know he might be on the "bad" list, but his background is so interesting. Eislyn(Reyna's sister) is really so sweet, but calculating. I enjoyed her and Thane's dialogue. The author did an amazing job with the imagery in this book. Everything was so detailed it was easy to fall into the scene. I love unexpected twists and while part of the ending I expected, I wasn't expecting how it took place. All in all, I found it very entertaining and I am very invested in continuing this series.
Favorite quotes:
"The truth may be twisted but never false."
"Who was she if she was not the enemy of the Air Court? What was her purpose of she no longer has that?"
"In a war-torn land, love was always a lie."
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Reviewed in the United States on March 4, 2020
★★★★★ 5
Great Read!!! Great story!!!
Format: Kindle
The series is long, but Ms. Wolfhart does a fantastic job of weaving this tale while bringing so much to the characters. Surprises and plot twists along the way to keep you intrigued. There is some graphic sex, but is no way the focal point. Grammar was excellent (a rare find with a lot of self publishers) with only a few noted errors. I rarely give 4 stars, let alone 5.
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Reviewed in the United States on April 30, 2021
★★★★★ 3
Sadly, DNF
Format: Kindle
I read this thru KU. I LOVED the synopsis. And then I began reading... and it was a DNF at 68% after picking it up and putting it down several times because I really loved the main female character.
*****SPOILERS*****
Pros:
The world is unique, intriguing and fun. The primary female character is bad-a** but not a b*tech or a mary sue. The primary female has depth. I really want to know what happens to her even tho it's been weeks and I don't remember her name. The villains to the point I read are pretty good -- an ever present threat of mysterious and possibly many culprits.
Cons:
Way, way too many points of view. I stopped counting at 7. It's the prime reason why I don't care about most of the characters or remember their names even when I like them. There's just too many points of view so almost none of the characters have enough book space for the author to properly develop them. This literally killed the book for me. Actually it killed my desire to read. For weeks.
The main male is more villain than hero. He agreed to marry the main female then locks her up & eschews her for her sister, all while bad mouthing her as unfit to rule when he never spent any time with her getting to know her. He is actually unfit to rule as he is blind to the woes of his own kingdom and starts off a peace mission to secure a ceasefire through marriage by murdering an inn full of people in her country for no real reason. Plus, he constantly makes promises he does not keep. And it's gross of him to pine for the sister behind the main female's back.
*****
As much as I really wanted to see what happened to the main female character, it wasn't enough for me to keep trying to slog thru this book. There was a lot of potential here that just fell short.
Hence, 3 stars.
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Reviewed in the United States on June 19, 2021
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