SKU: 81853634746

Gastric Organoid Cytokine Set, Mouse

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Description

Gastric Organoid Cytokine Set, MouseProduct Specification Species Mouse Endotoxin <0. 1EU g Physical Appearance Lyophilized powder Reconstitution Reconstitute at 0. 1 1 mg ml according to the size in ultrapure water after rapid centrifugation. Stability & Storage 12 months from date of receipt, lyophilized powder stored at 20 to 80. 3 months, 20 to 80 under sterile conditions after reconstitution. 1 week, 2 to 8 under sterile conditions after reconstitution. Please avoid repeated freeze

Product Specification


Species Mouse
Endotoxin <0.1EU/μg
Physical Appearance Lyophilized powder
Reconstitution

Reconstitute at 0.1-1 mg/ml according to the size in ultrapure water after rapid centrifugation.

Stability & Storage

·12 months from date of receipt, lyophilized powder stored at -20 to -80℃.
· 3 months, -20 to -80℃ under sterile conditions after reconstitution.
· 1 week, 2 to 8℃ under sterile conditions after reconstitution.
· Please avoid repeated freeze-thaw cycles.

Components

Protein

Reference dosage

100 ml system

1000 ml system

cytokine 1

100ng/ml

10μg

100μg

cytokine 2

100ng/ml

10μg

100μg

cytokine 3

50ng/ml

5μg

50μg

cytokine 4

100ng/ml

10μg

100μg

cytokine 5

100ng/ml

10μg

100μg

cytokine 6

100ng/ml

10μg

100μg


Protocol

Preparation of Complete Culture Medium for Mouse Gastric Organoids: Mix the basal culture medium, cytokines (1-6), and various additives in the predetermined proportions to prepare the complete culture medium.

1. Primary Culture
- (1) In the laminar flow cabinet, quickly open the abdominal cavity of the mouse, remove the stomach tissue, and place it in pre - cooled PBS (with penicillin, streptomycin, and primary antibiotics). Open the stomach, rinse the contents, and remove the outer layer of fat and blood vessels.
- (2) Collect the cleaned tissue blocks into a 1.5ml EP tube. Use a sterile fine - tipped ophthalmic surgical scissors to mince the tissue blocks as finely as possible (into a homogenate). The degree of tissue mincing is related to the digestion time. The finer the mincing, the shorter the digestion time. Transfer the minced tissue to a 15ml sterile centrifuge tube.
- (3) Add 10 times the volume of tissue digestion solution for digestion. Incubate at 37℃ with shaking for 10 - 30min. Observe the situation at any time during the digestion process. When a large number of cells are observed to leak out in the field of view and most of the cells are in the form of cell clusters, the digestion can be terminated (note: do not digest to the single - cell state).
- (4) In the tissue suspension confirmed to be digested, add fetal bovine serum (FBS) to a final concentration of 2 - 5% or bovine serum albumin (BSA) to a final concentration of 0.1%, and mix by pipetting.
- (5) Pass the tissue and cell suspension through a 70µm cell strainer. Centrifuge the filtered cell suspension at 1000rpm for 5min. After centrifugation, discard the supernatant.
- (6) If obvious red precipitate (red blood cells) is observed in the precipitate after centrifugation, add 2ml of red blood cell lysis solution, mix by pipetting, and leave it for 3min. Centrifuge at 1000rpm for 5min, and then discard the supernatant.
- (7) Resuspend the precipitate with an appropriate amount of basic culture medium or PBS.
- (8) Mix the matrix gel and crypts in a suitable proportion. For example, for a 24 - well cell culture plate, spot 25 - 30ul of the matrix gel mixture into each well for coating (operate at 4℃).
- (9) Place the coated culture plate in a 37℃ incubator for 20 - 30min to gel. Add an appropriate amount of mouse stomach organoid complete culture medium (at room temperature) for culture.

2. Organoid Passaging Culture
- (1) Aspirate the culture medium with a pipette, add 1 - 2ml of 4℃ PBS to each well, and leave it for 2min.
- (2) Gently pipette the matrix gel to collect it in a 15ml centrifuge tube. Adjust the volume with PBS to 10 - 14ml, and leave it at 4℃ for 20 - 30min. Centrifuge at 1000rpm for 5min to discard the liquid and retain the precipitate (group every 3 - 5 wells).
- (3) Resuspend the organoids with an appropriate amount of matrix gel, and spot 25 - 30ul of matrix gel into each well of a 24 - well cell culture plate. Place it in the incubator for 20 - 30min to gel, and then add an appropriate amount of mouse stomach organoid complete culture medium.

3. Organoid Cryopreservation
- (1) Aspirate the culture medium with a pipette, add 1 - 2ml of 4℃ PBS to each well, and leave it for 2min.
- (2) Gently pipette the matrix gel to collect it in a 15ml centrifuge tube. Adjust the volume with PBS to 10 - 14ml, and leave it at 4℃ for 20 - 30min. Centrifuge at 1000rpm for 5min to discard the liquid and retain the precipitate (group every 3 - 5 wells).
- (3) Add an appropriate amount of organoid cryopreservation solution, and gently pipette to resuspend. For example, for a 24 - well cell culture plate, cryopreserve 2 - 3 wells in one tube, with a volume of 1ml per tube.
- (4) Label the information properly, and then transfer it to liquid nitrogen for long - term storage after programmed cooling.

4. Organoid Thawing
- (1) Take 10ml of DMEM/F12 basic culture medium in a 15ml centrifuge tube.
- (2) Remove the cryopreserved organoids from the liquid nitrogen tank, and quickly place them in a 37℃ water bath to thaw.
- (3) During the water bath thawing process, gently shake the cryovial to ensure that the cryopreservation solution is completely thawed within 1 - 2min.
- (4) Quickly transfer the thawed organoids to a 15ml centrifuge tube, gently pipette 6 - 8 times with a pipette, centrifuge at 1000rpm for 5min, and then remove the supernatant to collect the organoid precipitate.
- (5) Resuspend with matrix gel, spot 25 - 30ul of matrix gel into each well of a 24 - well cell culture plate, place it in the incubator for 20 - 30min to gel, and then add an appropriate amount of mouse stomach organoid complete culture medium.

Guidelines

Avoid vortex oscillation; Store in aliquots to reduce the number of freeze-thaw cycles

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SKU: 81853634746

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bellconnor12
Lexington, US
★★★★★ 5
Excellent
Format: Kindle
An excellent work on the essential shape of pastoral work. A very convicting and important read for those who work in ministry- prayer, scripture, and spiritual direction will never be demanded of us, but are non-negotiable in order to maintain our call.
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Reviewed in the United States on August 3, 2024
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James Grimes
Draper, US
★★★★★ 5
A must read for any pastor or spiritual leader
Format: Paperback
This book is profoundly insightful and easy to read and understand. It is not heavily theological (and does not need to be). It is practical. I have purchased an additional copy to give my pastor for his birthday. If he has already read it, he can pass it on. EVERY pastor needs to read this and practice these tips, which many do not receive in seminary. THIS is the type of pastor I want and need (and my pastor does embody much of what Peterson describes). I think pastors should read this every year or two to reassess their progress toward keeping these goals always in mind and in practice as much as possible.
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Reviewed in the United States on June 26, 2019
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Michael F. Nelson
Birmingham, US
★★★★★ 4
A still timely writing for all pastors
Format: Paperback
Although first published 25 years ago, Peterson addresses a matter that is still timely, the need for all pastors to commit themselves to prayer, reading scripture and spiritual direction. These, Peterson asserts, are an essential foundation for all other aspects of ministry. Peterson's premise, that many pastors have abdicated their primary calling to these three practices, is still very relavent to the current state of pastoral ministry. Its content will be useful for new and veteran pastors alike.
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Reviewed in the United States on January 14, 2013
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C S Berry
Draper, US
★★★★★ 5
Transformative if you are willing.
Format: Kindle
The triangle of pastoral ministry - Scripture - Prayer - Spiritual Direction is reckoned as sever lack in our pastoral lives. Peterson is a needed sage in the realm of modern evangelicalism.
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Reviewed in the United States on March 13, 2024
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G. Sanders
Massapequa, US
★★★★★ 5
A Personally Challengiing Read
Don’t expect Rosaria Butterfield to coddle sin. She acknowledges the Biblical truth that we are at war with it. It is evil. Lies are rampant with it. The Bible’s solution is to kill it, nailing it to the cross of Christ. If we don’t, it will do great and lasting harm. That is the background premise of this book. This book is for believers in Jesus Christ, especially those who identify as Evangelicals. Others may benefit by reading its pages, but I suspect that most of them will not be able to persevere through it because today’s culture is just not willing to sit through this much proclamation of Bible truth. They will react, not respond. Believers will be convicted, encouraged, and edified. Butterfield’s style is plain, but today’s culture requires her to address and to use terminology that may be challenging to many readers. It was for me. Add a level of abstraction for some challenging spiritual truths. Add the fact that she is an intellectual dealing with cultural and theological subjects. It is mostly college level reading, except for the story parts. Butterfield has credentials. A professor of English at Syracuse University, she was ten years in the Lesbian community with a mate. She was found by Christ, began to grow in Him, was discipled by faithful church women, met a wonderful man, was married to him--a Presbyterian pastor—became a mother, and began to minister to college students from a Christian perspective. She tells some of her story and the stories of others in the pages of the book. It is fascinating reading. The book is organized around the five lies that Butterfield has chosen to expose, with two to four chapters dealing with each. She acknowledges that there are others that could be exposed as well. The last one, about our culture's attitude toward modesty, was the most surprising to me. The book also has a Foreword by Kevin DeYoung, a Preface, and an Introduction. Don’t skip them. The Foreword is nothing short of a good sermon that should be shared in every church in America. In the Preface Butterfield spills the beans, revealing exactly where she stands on key topics and to whom she is addressing this volume. The Introduction is engaging and lengthy and pretty much summarizes the rest of the book. Along the way Butterfield deals with homosexuality, changing gender, male leadership in the home and in church, the bedrock importance of repentance, progressive sanctification, intersectionality, “gay Christianity” (Side A and Side B), feelings and truth, empathy and sympathy, submission of the wife, feminism, inerrancy, envy and biblical contentment, suffering, modesty and exhibitionism, and even worship. She quotes scripture, sometimes at length, and she gives copious Bible references. She also has footnotes, some of which are as interesting as the text. She names and uses or responds to contemporary authors on the topics at hand. She gives solid directions for those dealing with contemporary issues. She also has suggestions for dealing with family members beset by these matters including a question-and-answer section. Page 301. I recommend this book for all Christian leaders and for believers who want to understand contemporary challenges for the church from our modern culture.
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Reviewed in the United States on May 27, 2024

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