First , the principle
Matrigel is a matrix component extracted from mouse EHS sarcoma, containing LN, type IV collagen, contact protein and heparin sulfate polysaccharide, deposited on a polyvinylpyrrolidone-free polycarbonate filter and reconstituted in DMEM medium. A film structure is formed which is very similar to the structure of the natural matrix film.
The membrane pore size is generally 8um, and the membrane pores are covered by Matrigel. The cells cannot pass freely. The hydrolase must be secreted and can be passed through the Martrigel-coated membrane through deformation movement, which is similar to the in vivo condition.
The Martrigel-coated membrane is placed between the upper chamber of the Blind Well chamber or the MICS chamber, with the Martrigel surface facing the upper chamber, and a chemotactic agent added to the lower chamber, such as a certain concentration of LN, FN or mouse 3T3. Liquid or human testicular epithelial fibroblast culture medium, the upper chamber is added with resuspended tumor cells, and the invasive cells begin to undergo membrane movement under the induction of a chemotactic agent. The time taken for the cell to penetrate the membrane was related to the amount of Martrigel. The 25 ug of Martrigell was selected and the results were more suitable after 16 hours. Most of the cells that penetrated the filter membrane adhered to the lower surface of the filter membrane, and the cells on the upper surface were wiped off with a cotton swab, and then the filter was fixed with ethanol, stained with PE, and the number of cells passing through Martrigel was observed under a light microscope. In addition, the Transwell chamber can also be used to reconstruct the invasion of the membrane. This method is to place 30ug Martrigel on the filter of the Transwell chamber hanging basket, and observe the result after adding the cells for 72 hours. It is worth noting that after the cells were cultured in the TransWll chamber for 72 hours, a considerable number of cells that passed through the filter membrane no longer adhered to the lower surface of the filter membrane, but fell off into the human lower chamber solution. Therefore, this part of the cells should be taken into account when counting the number of cells passing through the matrix membrane. The ability of tumor cells to cross the reconstituted stromal membrane has a good correlation with its ability to invade and metastasize in vivo. The reconstituted matrix membrane model can be used to screen anti-invasive drugs.
In the above analysis, if the Martrigel is not placed on the membrane and the 8um pore filter is placed directly between the upper and lower chambers of the invasion chamber, the cells move through the membrane through deformation movement, and the model is used to analyze the cell motility and drug-to-cell. The impact of athletic ability. In addition, the presence of LN or FN in the lower chamber or the deposition of LN or FN on the lower surface of the filter can analyze the effect of the drug on the chemotaxis or firmness of the tumor cells.
Second , the material
1, Matrigel Matrigel (Weiglas Biotechnology (Beijing) Co., Ltd.), 10mg / ml, 5ml, dispensed into 0.5ml / 10 EP tubes; add 0.5ml of DMEM when used;
Matrivgel maintains a liquid state on ice and quickly condenses into a gel at room temperature. Before use, it should be transferred from -20 °C to 4 °C until it is naturally dissolved (such as overnight) to avoid repeated freezing and thawing. Test tubes that need to be in contact with Matrivgel, pipette tips, etc. should be pre-cooled at 4 ° C; pay attention to aseptic operation;
2, 24-transwell (Coster);
3. Crystal violet dye solution: crystal violet is formulated into 0.5% mother liquor with methanol, and the concentration is 0.1%, which is diluted with 1:4 after PBS;
4, 33% acetic acid;
Third , the experimental steps
1. Solution preparation:
(1) Dissolving DMEM 500ml;
NE---A solution (1μg/μl, 1mg/ml)
Mother liquor 0.1ml (5mg) + ddH2O up to 5 ml; filter and disinfect, store at -20 °C.
NE-DMEM (-6M)
NE---A solution (1μg/μl, 1mg/ml) 20.5μl
DMEM UP TO 100 ml
Filter disinfection, storage at 4 ° C
(2) NE+CGRP-DMEM (-6M, 100ng)
NE---A solution (1μg/μl, 1mg/ml) 20.5μl
CGRP-storage solution 50 μl
DMEM UP TO 100 ml
Filter disinfection, storage at 4 ° C
(3) NE+IFN-DMEM (-6M, 50ng)
NE---A solution (1μg/μl, 1mg/ml) 20.5μl
IFN-γ (25ng/μl) 25 μl
DMEM UP TO 100 ml
Filter disinfection, storage at 4 ° C
(4) Low serum DMEM medium (upper room)
(5) 20% FBS-DMEM medium (lower chamber)
2 , preparation
(1) Sol, overnight at 4 ° C (Thaw Matrigel at 4 ° C overnight.)
(2) Matrigel is easy to gel at room temperature, so steps 2 and 3 use test tubes and tips are pre-cooled at -20 C before the test. (Matrigel tends to form gel very quickly at room temerature, therefore, pipets and tips using in steps 2 and 3 have to be chilled at prior to experiements.)
3 , coated base film (operating on ice)
(1) Dilute Matrigel gel (10 mg/ml to 5 mg/ml) with serum-free cold cell culture medium DMEM (Dilute Matrigel (10 mg/ml to 5 mg/ml) in serum free-cold cell culture media (RPMI1640, EMEM, DMEM, etc).)
(2) 100 ul of the diluted matrigel into the upper chamber of 24-well transwell
(3) Incubate the transwell at 37 ° C for at least 4-5 h (Incubate the transwell at 37 Cat at least 4 to 5 h for gelling.)
4 , hydrated basement membrane
Gently wash gelled matrigel with warmed serum free-culture media.
5 , prepare cell suspension and chamber
(1) Digestion method to obtain cells from a cell culture flask; (Harvest cells from tissue culture flasks by Trypsin/EDTA;)
(2) Wash the cells 3 times with culture media (RPMI1640, EMEM, DMEM etc) containing 1% FBS.)
(3) Resuspend cells, 5×105 cells/ml, 1% FBS (Resuspend the cells in media containing 1% FBS at a density of 5×105 cells/ml.)
(4) Put 200 ul of the cell suspension onto the matrigel.
(5) 600 ul of cell culture medium containing 5 ug/ml fibronectin as a lower chamber of the transwell is filled with 600 ul of culture media containing, as an adhesive subtrate.
6, incubation, 37 ℃, 20 to 24 h (Incubate at 37C for 20 to 24 h.
7 , staining and counting
(1) Scrape off noninvaded cells on the top of the transwell with a cotton swab
(2) Remove transwells from 24-well plates and stained with Diff-Quick solution.
(3) 500 μl of 0.1% crystal violet was added to a 24-well plate, the chamber was placed therein, the membrane was immersed in the medium, and taken out at 37 ° C for 30 min, and washed with PBS.
(4) Take 4 fields of view on the diameter, photograph and count.
(5) 500 μl of 33% acetic acid was added to a 24-well plate, the chamber was placed therein, the membrane was immersed, shaken for 10 min, and fully dissolved. The chamber was taken out, and the 24-well plate was measured at 570 nm on a microplate reader to indirectly reflect the number of cells.
Tip: Be sure to dry before taking pictures. Place the chamber on the slide while taking pictures, and observe and take pictures under an inverted microscope. Economic, practical and convenient.
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About Esco Medical
Esco Medical has been serving the Dental Industry for nearly 20 years from China. Over the years it has been known by a few names, but has always pursued a single goal of serving our customers
In 2000, [Esco Medical" was founded in Suzhou China. HB Dental specialized in dental chair-mounted unit.
[Esco Medical"has accumulated rich export experience since it started export orders in 2002 and got ISO9001 Certified.
By providing financial stability and operational expertise, it has allowed HB Dental to remain focused on its values and work towards building the company`s core competencies.
The Esco Medical team has built a reputation of quality and integrity in our local community as well as the Dental and Health Care Industries. We are proud of achieving an A+ rating with the Better Business China, and continue to work hard to ensure our customers receive the best value and service available.
Esco Medical prides itself on providing a full & efficient service regarding dental equipment and equipment as well as dental furniture to customers around the world.
Supporting it are our experienced sales, product consultants, service engineers, especially an ISO9001:13485 certified factory with strong ability in researching & creation, as well as the close & sustainable cooperation with many famous factories.
As our core-competitiveness, a research & development team led by a senior engineer who has 30 years experience in manufacturing dental equipments keeps our innovation shouldering with the top technology in global dental field. Customer`s satisfaction is always put in the most important position in our researching, manufacturing and sales as well as after -sales.
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