Enhanced cell adhesion kit-3(ECAK-3)

Increase your cell productivity potency by 10 times, 100 times
Simple preliminary experiments yield results

WI- 38, MRC- 5, FRhL- 2, 2BS, KMB17, human diploid cells (HDCs), BHK-21, HEK-293, walvax-2 BV2 A172

Enhanced Cell Adhesion Reagent Instruction Manual Product Description:
Although all kinds of cell culture consumables have been processed to a certain extent and can support the culture of most of the adherent cells, some cells need to be adhered to the wall but are difficult to be adhered to the wall, and need to be adhered to the wall to achieve adherence culture, such as the chicken liver cancer cell line LMH, etc.; there are also some cells, such as 293T, etc., which are not adherent to the wall, resulting in the subsequent process of the test, such as transfection and/or viral infections, etc., due to the long incubation time, the cells will be easily detached. Some cells such as 293T are not tightly adherent to the wall, causing the cells to detach easily due to the long culture time. These problems have always been troubling the majority of researchers engaged in related research. This reagent has been optimised through repeated tests of various formulas, and is made of more than ten kinds of ingredients, such as cell culture grade inorganic salts, many kinds of synthetic high-purity small molecule proteins and many kinds of protective agents, etc. It has the advantages of simple operation, tight cell adhesion, non-shedding of cells, and non-toxicity to cells after treatment.

Remarks:
Alternative common methods recommended: Fibronectin coating, gelatin plate, cell culture equipment, tissue engineering, Transwell invasion experiment (wall-promoting part, nutritional part is not included), FN-coated culture plate, Matrigel coating (wall-promoting part, nutritional part is not included), polylysine-coated culture plate, collagen type I protein pre-coated culture plate, human fibronectin coating plate, human fibronectin coating plate, collagen type I protein pre-coating plate, collagen type I protein pre-coating plate, collagen type I protein pre-coating plate, human fibronectin coating plate. The users can complete the related experimental problems together with the users of the plates, human fibronectin-encapsulated plates, talen lentivirus encapsulated with 293T cells, cell culture materials and so on.
Not recommended for users:
We don't recommend to use the cells cultured by domestic reagents and consumables with unknown cell sources, and the cells not adhering to the wall caused by serum quality are not suitable for our products.

What is the difference between Enhanced Cell Adhesion Reagent and other reagents such as Gelatin, Fibronectin, Polylysine, etc.?
The difference between gelatin and polylysine and these two reagents is that our product is easy to use and has very little toxicity. Several cell lines have already been tested, completely reversing the ATCC gelatin protocol.

The price of Fibronectin is very high, so we have a big advantage.
Microcarrier encapsulation, experiments have been done, and need pictures can be emailed to the letter.

Solve the problems of LMH cell mass culture process, such as cell hard to attach to the wall, slow value-added, easy to agglomerate, etc., the use of unique wall-attachment reagents, with the optimisation of the culture method, so that the cells can be attached to the wall in 3-5 hours to grow, 36-48 hours can be passed on to the culture, the cell attached to the wall of the culture for a long period of time, the cells can be grown full of maintenance of 7 days, and still maintain a good cellular morphology. 4 adenovirus proliferation of high titer, TCID50 up to 8.0 The immunological effect of the vaccine was precise, and the immune group was 100 per cent immunised. The immunity effect of the vaccine was exact, 100% protection in the immunised group, and all deaths in the control group after the attack, and typical symptoms of pericardial effusion-hepatitis syndrome appeared after autopsy.

LMH cells completed 60-70 generations of cell culture, cell recovery and growth without any attenuation changes. Other reagent relevance and relevance of serum-free, good choice of culture reagents, so that the cells without degradation to achieve 100% passaging, serum-free temporarily can not replace the serum programme, it is recommended that a variety of serum-free programme cells with serum programme preservation of the seed, for example: cho sf9 sf21 and so on.
2018 cell verification: lncap (tumour formation experiments have been completed, the article has been issued,A172,293T confocal experiments, jurkat transfection before wall domestication. lmh cells more than 150 generations of no change, the subsequent experiments have no effect)
2019 Cell validation: LMH cells passaging experiments TCID50 six point zero or more, neural-like cell lines, stem cells growing against the wall. Suspension cells transfected and domesticated into adherent cells in the experiment.
Case: LNCAP cell culture has great advantages, and it can replace the foreign encapsulated culture plate completely. Industrial application cases see attached, foreign journals in June can be queried and cited!
Article labelling.
BeiJing XiGong biological technology co.
Enhanced cell adhesion kit-3(ECAK-3)
CAT:C028
After-sales service and sales service is complete, welcome to write to us for more details.

20200915 Update Note: Transwell invasion assay\Matrigel coating (pro-adhesion part, nutrient part is not included)
After two years after the launch of the product and the use of user statistics and summary: 1ml of reagents can be diluted to 10ml-50ml programme have good results. Recommend our original imported PBS dilution. Subsequent to the user's request for dilution, we can match the imported PBS ready-to-use reagents used.
Case cell pictures (product information) two 293 case cells adenovirus infection titre difference of more than 10 times.

Cell factory application users can discuss the details of the culture part, we always statistics in the use of common problems.

20200919 update statistics application supplement: case of several strains of cells after several years of culture statistics application concluded that the cell recovery step of the reagent has to promote the cell bed, is conducive to cell proliferation and growth. The case cells will be listed in succession.
After promoting wall attachment, monoclonal, fluorescent cell screening also has a promotional effect.

20201110 update statistics application supplement: resuscitated cells when the bed time faster, ready to apply a few famous refractory cells, BEAS2B, INS-1, HK-2, THLE-3 and other primary, all have the characteristics of the bed is not firm. ATCC source, the cell growth does not go well at the critical time period, can be separated from a small number of cells to test. For similar cases, please write to us. Successfully rescued user knockout mouse-derived MEF precious strain.

20201120 update statistics application supplement:Beijing through the gift of good quality 293FT, HELA two strains, statistics laboratory culture habits on the cell impact, reagents part of the not to say, the technical part, do not fully understand the digestive cell trypsin chemical action, to avoid mechanical force on the cell damage, that is, the reagents are better, blowing the cell after the cell broken, surviving the rest are smaller size cells, these The habit of raising cells, subsequent transfection, virus packaging, infection and other experimental results decline year by year. And many laboratory follow-up experiments have more fixed steps, affecting the final experimental results, welcome to discuss with all the newbies, post-transfection digestion and other details of the steps, mailbox or add my WeChat, specify: digestion step discussion.

20201201 update statistics application supplement: drug-resistant strains of screening applications, to avoid the drift of the cells after the addition of drugs to save time, increase the IC50 feasible (before adding the drug cell state adjustment is very important, welcome to discuss, inform the details of the trypsin and promote the wall can be sent to the trial)
Simple elaboration: drug concentration, large dose concentration, the lowest concentration, gradient pull out, any concentration group in the middle, add the drug three times screening, the cells can survive after the generation of drug-resistant strains. If you need monoclonal, wait for the cells to collect, add agar to fix the cells, and then punch out the target cells.

20201210 Rescue a strain of MEF from a precious gene-edited mouse source

20210104 Immunofluorescence application, good cell stretch, clear cellular component devices, cells withstand tossing, newer labs with newer imaging equipment welcome

20210630
Cellular pro-anchoring applications: screening of resistant strains, repeated dosing, cryopreservation and resuscitation. Rescue cells, confocal, 293 series characteristics, SV40 transfection of semi-suspension and semi-adherent characteristics or more difficult to raise cells, primary epithelial screening to replace collagen, neurological primary cell lineage class to replace the adhesion protein, protein high-yield serum-containing cell factory programme has been industrial applications.
Case: 293 promotes wall affixation with and without, low concentration trypsin digestion time difference of 20 minutes. Different concentration gradients can be diluted to filter out the walling strength required for experimental purposes. After the use of low concentration long-term pro-adhesion wall, the cell trend is good, imported reagents and consumables
Long-term passaging after the user to do cell karyotype analysis letter to inform, immunosuspension or apoptosis users have this test requirements can be discussed, reverse cell domestication we have some theory, the case is not a lot. Regular cell bank teacher to have time to test, the product is all free to test, no quantity limit.

20211013: immune cells, primary cells transfected with retroviruses, in our promotion of wall reagents for the development of new applications, the effect of a clear technological breakthroughs, POTOCOL is still in the optimisation, the key issues can be discussed!

20210910: drug-resistant screening focus, with drug transmission, application of drug-resistant strains class screening is the same, simple and easy, began to undertake 293 stable strain clone screening trial operation, short time (14 days to complete the G418 screening of 800ug / ml Figure 1 is the monoclonal growth of amplification)
Our advantage should be anchored in the recovery of the cell after freezing and storage, promote wall affixation advantage is greater, the transfection reagent credit is not small, Figure 4 is the transfection efficiency is very high after the G418 dosing of live and dead cells presented.
Technical discussion details to be informed first, individual details.
Primary epithelial lineage construction is the final purpose. Note: G418 concentration is screened according to the status of your own cells.

Screening antibiotics
G418, Geneticin Zeocin Puromycin Blasticidin Hygromycin B
The above try to use the big manufacturers, other brands are recommended to pay attention to the following parameters (compared with the big manufacturers, to avoid different conditions affect the subsequent experiments)
Purity (HPLC)
Potency claimed (µg/mg)
Potency upon retest (µg/mg)
ED50 Assay (µg/ml) ED50 assay (µg/ml)

Pre-transfection status adjustment
90% of cells - cell activity can be easily determined using Taipan Blue staining.
We recommend Optimised Trypsin for pre-transfection.
Dilute DNA in DNA/RNAase free water or TE.
Concentration of DNA
It is recommended to spread the cells into 2 different densities to ensure high transfection efficiency.
DNA can be prepared as early as 2 hours in advance, but not too early; on the day of transfection, spread the cells and keep the cell density at 70-90% confluence during transfection
After mixing equal volumes of diluted transfection reagents and DNA solution, mix the solution by slowly blowing up and down, either by flicking the bottom of the tube or by rapid vortexing.
Add the complex dropwise to the medium and then gently shake the plate. Do not violently disperse the liposome/DNA complexes in the wells, as this will displace the cells. Transfection efficiency was assessed using plasmids containing reporter genes like GFP or LacZ as positive controls.

293T is resistant to FT and is not suitable for the above screen, 293 is suitable for

20211101: also welcome to discuss cooperation with the production of biological products with GMP qualification Beijing nearby companies, the current application cases have pilot production of related protein users (in the freeze-drying protective agent encountered bottlenecks, also welcome with the domestic freeze-drying protective agent R & D and production of related cooperation with the relevant - related to the study of the technology teacher is welcome to join - foreign technology is quite mature - our upstream process has been a good result, the protein production can only be negative twenty preservation, but also the production of the protein is not suitable for the above screening, 293 is suitable for the above. (Protein production can only be negative twenty preservation, quite embarrassing)

20221013: rescue G418 screening HUH7 strain, after recovery, the cells are not anchored, the reagent on the rescue success, the drug-resistant strains of rescue analysis, the addition of drug cell damage, and the difference between the conventional growth is very big (easy to form a piece after the addition of drug, into a ball - also due to detailed statistics of this type of cell characteristics, epithelial, epithelioid, glial growth characteristics, all have this type of characteristics, the method of statistics is to solve the problem of this type of growth characteristics, the recovery of cells into a piece after the freeze storage, and the growth of cells into a ball. (Growth characteristics, cell viability after resuscitation is very illustrative of the problem, the subsequent solution of this type of optimisation of cell adjustment before transfection also significantly improve the efficiency of transfection, in short, are to solve the characteristics of these clumps into pieces to enhance the quality of the cells have significantly improved) suggested that teachers do drug-resistant strains when the seed preservation reference, clumps into pieces of cells: HEPG2, THP-1, RAW264.7, JURKAT, lymphoma, BV2, SV40, BV2, JURKAT, lymphoma, lymphoma, lymphoma, lymphoma, and so on. BV2, SV40-Large T antigen transfection and lineage building, there are many lineage building labs in China, all aspects of lineage building can be discussed, in vitro processing, culture (adding factors, promoting anchoring), transfection - reagent-based (primary, stem cells, immune class difficult to transfect the problem of statistics), seed preservation and so on. Those who know about transfection of immune cells for infection requiring cells, protein anchoring can write to discuss.

20221111 Progenitor keywords can be discussed in statistics:
Isolate preservation digestion processing: equipment + reagents + statistical experience discussion
Cytoplasmic basophilic: medium selected techniques, literature, experience and other comprehensive considerations discussed (serum-free mature strains, difficult to raise strains statistics).
Adhesion: collagen, gelatin, adhesion protein, albumin, polyclonal, Matrigel matrix gel, pro-adhesion reagents, etc., combined with the problem of clumping into pieces, the later cells are not ideal, the detailed work of the statistics and refer to the different places with these products. And a variety of experimental fine work to solve the idea.
Gas-phase flow in the original generation after screening statistics began, conventional liquid-phase flow screening after building lineage purpose of rescue began statistics.

20230412
Enhanced affixation reagents for difficult-to-transfect cells method reference
(primary immune, primary normal, glial,)
Enhanced wall affixation treatment culture plate after PBS washing
1. Melt the retrovirus preservation solution (-80 ℃) in a 37 ℃ water bath.
2. Add the virus solution into the coated 24-well plate, 300 μl per well.
3. Leave at 32 ℃ for 4 hours.
4. Aspirate out the virus reservoir.
5. Add 400 μl of the cell suspension to be transfected into each well (the usual cell concentration is 1 × 105/ml).
For DNA and RNA transfection users, the recommended dosage ratios are optimised after referring to GIBCO-POTOCOL. Please contact POTOCOL for further information.
Trial sent in, the use of requirements, culture system all imported (preferably GIBCO system, the cells can be passed on normally with a high success rate) until 1 October 2023, the experiments are satisfied with the payment as far as possible, the cost is too high!
Inform the details of the cells (we have a relevant summary of the treatment after isolation, we can discuss the details)
The shipping cost is higher for foreign users, the price is tentatively set at $600.

20230720: high titer research, production is our company is now more mature, the application of homologous cell cases of high feasibility, smooth, easy to repeat, the core is the most basic cell state and quality, plus auxiliary reagents and cell maintenance culture combined, our company began to focus on the case of the source of cell quality assessment, technology, methods, reagents, all-round cooperation with the user to complete the goal of high titer needs.
Cells: 293FT (users with high requirements for cells can specify, share the ATCC direct source of the cell line, serum and pro-adhesion reagents users to share free of charge), the technical reserve prediction, the titer to reach the conventional parameters of 50 times, and strive for a hundred times.
Culture system: fine work to provide all the reagents, backup system and THERMO, Concord cell bank culture system. Three manufacturers programme seed preservation verification purposes, but also for industrial users of domestic system backup to provide relevant verification cell verification parameters.
Stealing the discussion ha: serum-free R & D we have a great advantage, a variety of details of the ingredients can be long-term verification, and strive to get the most cattle technology core. Serum-containing basic technology reserves are sufficient. It is also our advantage. (The above are all serum-containing programmes)
High-titre case cell discussion: the original generation to build a variety of liver POTOCOL difficulties can be discussed, it is recommended to start the mouse, chicken, duck easier and feasible, the subsequent import of refractory cells to do something about it.

20240410: Phagocytosis industrial application case reference: Sometimes it is not the isolation of the virus, is the cell maintenance culture time is not enough, especially difficult to isolate, new viruses, hotspot applications, etc., from zero to one is the top priority. Cell maintenance culture is the key, after five years of adenovirus, lysovirus, cell seed bank level reagents, cell factory applications and other related technology reserves redundant industrial reagent providers application characteristics, we promote wall-attached play a key role.
Case cells: LMH, maintenance culture on the tenth day of phagocytosis, the user has completed the monoclonal seeding, there is a related need for users to discuss the difficulties in-depth, recommended reagents and consumables, manufacturers of models, culture characteristics, purpose requirements, inform the details, we give reference to the recommendations, there are foreign vaccine manufacturers and users of the business, we can send a trial and solutions. We can also do third party verification. The cell factory contains the serum programme as the basis.

20240424.
Fine-tuning stable transducer strains to construct pain point advantages
Ultra-dose screening of stable quasi strains
1: Higher than normal dosing 2: Passage with drug 3: Resuscitation with drug
Industrial Advantage: After screening of stable transplants, ordinary culture conditions (reduce cost), routine laboratory (high feasibility), no need to add expensive equipment, the purpose of the protein expression 14 days of culture (cells to maintain the advantages of the culture state), can be perfect to complete the serum-containing serum-free a variety of needs. Laboratory experts are welcome to participate, we docking pharmaceutical companies joint development and application (fast cash), a variety of difficulties and pain points welcome questions. All kinds of serum-free culture medium, transfection reagents free of charge. (There is a foreign demand for users, high commission)

20240515
Application case one, the upstream development of the product has been mature, ready to transform the production.
The advantage of this reagent is to maintain the culture, no damage to the cells, extend the application of industrial vaccine production (lysin production of intravesicular products), exosome maintenance culture, supernatant secretion of various needs (umbilical cord blood and umbilical cord supernatant secretion engineering applications).
Detailed parameters of the industrial case: The production of avian adenovirus vaccine using LMH cells is usually around 107.0/0.1ml, which greatly limits the production capacity. After the application of this reagent, the TCID50 potency of avian adenovirus type 4 is more than 108.5/0.1ml (which can greatly shorten the wall attachment time of LMH cells, and the cell morphology is still good after 192h of cell culture, which is easy to replicate and proliferate avian adenovirus virus, as well as the subsequent determination of the virulence value. 4 avian adenovirus type 4 has TCID50 potency of more than 108.5/0.1ml, and the whole virus culture solution of 1ml can be formulated to produce avian adenovirus vaccine. The TCID50 potency of avian adenovirus type 4 is more than 108.5/0.1 ml. Currently, 1 ml of the whole virus culture solution can be used to prepare vaccine for 200 birds, which can protect SPF chickens 100% against the strong virus of avian adenovirus type 4 (100% death after 72h of virus attack in the unimmunised group). The results of this test have been verified several times in cell factories by different specialists of different manufacturers.
The above reagents and processes are mature, and we would like to enquire about the export requirements, this level of technology can be verified by a third party at any time (no substitutions, domestic or foreign), contact us by email.
Application case two, attached case picture
Phagocytosis industrial application case reference: Sometimes it is not the isolation of the virus, it is the cell maintenance culture time is not enough, especially difficult to isolate, new viruses, hot spot applications, etc., from zero to one is the most important thing. Cell maintenance culture is the key, after five years of adenovirus, lysovirus, cell seed bank level reagents, cell factory applications and other related technology reserves redundant industrial reagent providers application characteristics, we promote wall-attached play a key role.
Case cells: LMH, maintenance culture on the tenth day of phagocytosis, the user has completed the monoclonal seeding, there is a related need for users to discuss the difficulties in-depth, recommended reagents and consumables, manufacturers of models, culture characteristics, purpose requirements, inform the details, we give reference to the recommendations, there are foreign vaccine manufacturers and users of the business, we can send a trial and solutions. We can also do third party verification. The cell factory contains serum programme as the basis. Attached is a picture of 10-day phagocytosis for reference.
Involved points: trypsin digestion, maintenance medium application, promotion of anchoring, bottle culture to control PH changes, etc., specific problems specific analysis.

Application Case Three.
Pain Point Advantages of Stable Transformant Strain Construction for Production
Overdose screening of stable transplants 1: Higher than normal dosing 2: Transfection with drug 3: Resuscitation with drug
Industrial Advantage: After screening of stable transplants, normal culture conditions (reduce cost), regular laboratory (high feasibility), no need to add expensive equipment, the purpose of the protein expression 14 days of culture (cell maintenance culture state advantage), can perfectly complete the serum-containing serum-free a variety of needs. Laboratory experts are welcome to participate, we docking pharmaceutical companies joint development and application (fast cash), a variety of difficulties and pain points welcome questions. All kinds of serum-free and transfection reagents are free of charge.
Application case four, domestic lysoma virus R & D production based on 293 cells has been more than four years of smooth orders, more than five users.
Application case five: the scientific research household rescue cell more, the case is not one by one, precious cells (resuscitated cells are not anchored) other protein interactions, colocalisation, the original instead of generative collagenase advantage, tumour instability group cell lines, exosomes, cell product research, production, etc. can be sent to the trial, such as in-depth development of the difficult points pain points upstream process, it is recommended to inform the name of the cell, the amount of culture needs, the name of the product, the purification of the current! We are still weak, cell yield, generation, maintain culture advantage. Seed bank level reagents are more feasible, and overall cost reduction. This is our biggest advantage.

Seed bank to build a line to build the library, the details of the statistical content of more: also welcome the domestic medium, serum manufacturers to participate in the long cycle, the cost is high, can be successful to build a line of great significance. Welcome foreign trade exporters to introduce foreign users.
Reagent development related to technical reserves in the statistics of pain points:
Containing serum: to ensure the feasibility of building a high system buffer capacity, high fault tolerance.
Serum-free: in the serum-containing cells sufficient status quo, the development of relatively easy. Serum-free cell line problem analysis has positive significance.
Black point: common problems of domestic lineage construction, detailing can be from different grades of medium, serum, trypsin, resuscitation viability after freezing. Discussion of each pain point
Slow growth: common problems of domestic lineage construction, agglomeration into pieces, treatment after successful rescue, etc. According to the characteristics of cell tissues, lineage common problems analysis and give reference suggestions.
Tumour instability rescue: common laboratory common problems, fine work to give reference to the program, the statistics began in 2023, the effect is expected to be ideal, optimized, a 25 culture flask inoculation of several animals should not be too difficult, including PDX tumour and other pain points.
(After the final rescue, supporting the digestion of non-destructive trypsin is also critical. (Specific cell specific analysis)

20240529
Statistical references related to other applications:
Ultra-long time maintenance culture, no damage to cells.
Complete control of cell growth (can be completely and precisely control the cell experimental requirements of long, stop, maintenance), extended application of cell products, industrial vaccine production (lysosomal protein production of intra- and extra-vesicular products), exosomes maintenance culture, supernatant secretion of various types of needs (umbilical cord blood and umbilical cord supernatant secretion engineering applications).
Automated culture technology reserve reference, suspension and wall domestication (automated culture in the control of cell growth characteristics, wall suspension), a variety of needs of different related technology statistics and reserves.
Organoid application reference: pre-linkage culture higher feasibility of pain point discussion, no chemical interference after isolation, more feasible after machine interference, the premise that the culture is smooth after the monoclonal, the establishment of stable transplants, involving the points of easy to repeat the feasibility of the culture system, the purpose of lineage freezing resuscitation importance of the feasibility of the method of discussion.
Artificial meat: the first two-dimensional culture, cell volume is sufficient, in 3D culture, the laboratory does not have to go on the fermenter, cell factory is simple and easy to do, we have a user in hand to do the house transformed into a large-scale culture device, large capacity, low cost, low cost of the culture system to control the cost of simple and easy to do.
Vaccine applications: the above points are overcultured for the premise of simple and feasible, vaccine applications are relatively simple, maintenance culture containing serum programme cell buffer capacity is stronger, the head of the current several primary culture companies, do not pass on, maintenance culture for about a year (the original generation has not been built before the lineage, the disadvantage is that it can not pass on)
Maintenance culture (strong redundant reagent development): monoclonal expression, plaque-phagic protein crystals (crystals need to be high-titer protein), virus crystals, recombinant proteins, antibody production, monoclonal expression, veterinary vaccine applications, protein interactions, factor production
(There are two references for possible reasons for cell culture supernatant backfeeding, one is the secretion of growth-promoting factors; the other is the release of competitive signals that stimulate cell growth, which is why cells are too sparse to grow easily.)
Stable transplants to build the line advantage of the discussion of pain points: drug-resistant strains \ stable transplants to build, monoclonal expression, phagocytosis, primary cells, sv40, tHERT

Domestic cell culture system to build line manufacturing points of pain points can be discussed: purely domestic technology engineering cell line construction (engineering strains need to be verified for a long time for stability, pain points in advance of the problem and have a positive significance of the relevant technology reserves, strong redundancy reagent development, maintenance of the culture application, the manufacture of large incubators) and parallel to the imported system backup, long-term verification of the nuances.

20240625
Industrial case application I: the separation of avian symbiotic virus application: adeno, bean, circular single species purification separation, theoretical methods, pre-tests are more satisfactory, more case applications are still in the technical reserve, and the cooperation of the teachers to discuss the difficulties of the pain points, the Beijing laboratory can participate in the discussion of the subject, on the difficulties of the pain points of the problem, we do some statistics first.
Protein interactions on cell maintenance culture requirements of high users, can be discussed after the problem first, fine work to give some reference suggestions.

Domestic departmental laboratory, the growth time is too long, can also consider monoclonal or slow culture of two technical solutions, difficult to digest cells from the source is recommended to consider re-construction, the difficulties can be verified through similar problems of simple solutions of fine work, for example: difficult to digest (easy to solve the culture plate Pou serum), the fine work of the programme through the enhancement of tissue activity (not to be entangled in the digested activity is not high on the tissues or cells) the feasibility of high, the operation is simple, through simple operation steps, the fine work gives some reference suggestions. Simple operation, can be verified through simple operation steps, the back of monoclonal, stable transfer strains, cryopreservation and recovery, etc., are based on the feasibility of stable after the difficult pain points to verify the feasibility of the programme, in the application of the construction of the line or precious cells.

20240702
Application Supplementary Cases:
Suspension of immune cells, adherent wall domestication culture, colocalisation, CCK8, development of automated culture equipment, co-culture of organoid immune cells and tissue cells, etc.
Renewal of valuable strains in domestic laboratories: simple (monoclonal, de-mycoplasmaised cultures with long-term validation), self-constructed line culture optimisation to enhance growth rate and cell quality.
All kinds of tissue cells from the source of stromal gel to maintain the culture development of new technology cooperation (suitable for a large number of cell factory manufacturers, laboratories) long-term cooperation: suitable for adenoids, adeno-neurogenic coexistence, lymphomas, gliomas, sarcomas long-term cooperation from the beginning of the establishment of the line to complete the entire upstream process of development, the ordinary laboratory environment can be completed to the Chinese style, without the need for large-scale equipment and costly R & D investment, protein analysis, protein purification Domestic R & D producers pass by the message, we can only to the cell factory scale production of this step no BUG, no ability to do the whole. There are transformation laboratories can be in-depth discussion of pain points and difficulties.

20240901 Enhanced sticker reagent assisted recovery to verify the advantages: live rate and state are significantly improved, more than three years of frozen recovery can be compared to verify the live rate and cell stretch through a few days of recovery can be a significant difference (rescue cases), the advantages of this point in the recovery of the recovery can have a significant effect, the back of the laboratory conditions, in the usual culture is the same way to improve the proliferation of cells and the state of the transfection of the dead Cells more than one technical reserve, can also be used in the same way to make comparisons.
Note:
1: the flakes into clumps to be resolved before freezing, refer to our optimised trypsin characteristics.
2: high-quality serum is very critical in the freeze-storage resuscitation, it is also recommended to have the conditions of the group, the usual procurement of serum before verification as a key indicator, other cell verification serum can be discussed.
Seed bank-fine work complete set of freeze recovery reagent backup group
Recommended freezing method: DMSO10% + FBS90%
Higher requirements for freeze resuscitation, available cases of cells with fine work recommended procedure cooling box + cotton freeze cells, the domestic establishment of the line is recommended to have a backup group according to the above method.
The ultimate goal of cell culture - smooth cell state.
Commonly used cells for three years to do freezing resuscitation verification
Refractory cells 1-1.5 years to do cryopreservation recovery verification

20240915

Fine Work Redundant Reagent Development Product Advantage in Automated Transfection Applications - Enhanced Adhesion Reagent Use
Automation is used in many applications to reduce the variability caused by manual processing and to obtain reproducible results in high-throughput transfection analyses. High-throughput applications, such as knockout studies or target screening, often include cell transfection. Fine-tuned development of redundant reagents enhances the presentation of adherence reagents.

Poorly anchored, or immune cells can be assisted with Enhanced Adhesion Reagents, reverse domestication, and high throughput processing of cells for obvious advantages.
Routine automated generation and analysis of 3D cell cultures in Matrigel can be searched for relevant introductions, the fine work of another route, take the first to build the line, in the two-dimensional mass culture, after sufficient cell volume in the sphere culture (the advantage of uniform cell quality, maintenance will be cultured for a longer period of time, a slightly larger sphere is not defensible, supporting automation so that the cells according to the idea of what they are cultured) class of organs is currently a common problem of internal ulceration problems can be Increase the maintenance culture for a longer period of time. Artificial meat, exosome production is the same.
Suspension through the enhancement of wall-attachment reagents can be domesticated into wall culture - the purpose of fine lineage building (without the enhancement of wall-attachment, the cells are suspended primitive)
Difficult to turn the mainstream direction of the cell, reverse transfection method, to promote the wall after transfection efficiency greatly improved. Large factories have the method introduced, primary, immunological mainstream, with the method. (This method allows the immunosuspension to increase the transfection efficiency after affixing to the wall with auxiliary reagents).
The usual culture can be compared with the addition of pro-adhesion reagents after the state, refer to the advantages of cryopreservation rescue.

20240926

Introduction to scientific research and production applications
Diploid engineering cell application research, production, automated equipment + redundant reagents (technology) pure domestic technology production backup.
Primary cell production with generation limitations, greatly affecting the vaccine yield, fine work to enhance the paste wall method is and supporting, increase the number of days of cell maintenance culture, the corresponding increase in production capacity, the production of veterinary encephalitis B vaccine increased by 10 times, R & D to the Chinese style, can be turnkey docking, without the need for special and expensive equipment to add, serum-containing cell factories.
WI- 38, MRC- 5, FRhL- 2, 2BS, KMB17, human diploid cells (HDCs), BHK-21, HEK-293, walvax-2 BV2 A172 (the latter two strains are the purpose of the development of the new process of matrix gel)
Diploid engineering cell application research, production, automated equipment + redundant reagents (technology) purely domestic technology production backup. Expanding more application refinement and problem statistics
Redundant reagent development focus: conventional problems should be solved, including some difficult pain points should also have a breakthrough in the automated equipment, there will be super fault tolerance, automated soft reagents are expected to docking smooth with the hard equipment, the scale of culture extension applications, lineage building, etc. will be very smooth output of high-quality cells, proteins, proteins downstream R & D companies to be able to share technical information companies, to give some of the upstream R & D to add support.

20241009

Automation unified case cell welcome to participate, free cell seeds, reagents, requiring users to import consumables with the same standard, after the problem is solved accurately.
Such as: domestic imports of two systems, containing serum serum-free two systems, the accumulation of problems encountered after years of culture is not the same, the solution to the problem of ideas and methods are completely different, on the automation, unification, the alignment of the problem is relatively easy.
293, 293T
Medium, serum, trypsin, DMSO,PBS
Scientific research: the purpose is to do seed bank backup for domestic culture system, after the technology reaches the standard, we will add cell lines one after another, all of them are regular library sources. For example, if this cell line is related to lentivirus, adenovirus infection, and the purpose of establishing the line, we can discuss the application of high-titer amplification, establishing the line, and crystal research.
Production type: domestic culture system backup, provide cells, water reagents, imported consumables requirements, cases of cell seed bank satisfaction, suitable for diploid production applications. Directly to the Chinese style, application cases five years of uninterrupted production of relevant statistics.
Automated transfection, scale culture, related redundant reagent development.

Foreign cell culture vaccine production users to expand cooperation, fine work to enhance the paste wall reagents export, domestic validation laboratory, can be sent to trial.

20241115

Cell culture redundant reagents - microfluidic

Automation (machines, reagents, technology) combined with the completion of cell culture in the purpose of the needs, SOP needs to be accurate, the requirements for reagents are more demanding and accurate, some product development and exploratory exploration in the production of cell culture medium and refractory problems in the past ten years, the development of cell-promoting adherent reagents, trypsin, dissociation of cells in culture, existing products have made some achievements in industrial production, more than five years of application statistics and various applications, supporting microfluidic control culture to launch the concept of redundant reagents, The advantage of automated cell culture research is that the cell adherent suspension reverse acclimation culture, so that the automated equipment can complete the purpose requirements according to the requirements of the machine in suspension and adherent characteristics, through several years of experience statistics, the feasibility of containing serum is high, and the common culture conditions in each laboratory have basic routine habits to judge the results, which is simple and easy.
In view of the current hot research on organoids, artificial meat, cell line construction, and recombinant protein production, the serum-containing protocol is simple and easy to repeat, and after obtaining a large number of cells, it is relatively easy to verify serum-free, and sufficient cell volume can also have more opportunities to verify serum-free, which is relatively easy to achieve. Ultra-long-term maintenance culture, in cell interaction, protein interaction, exosome production, crystal research, protein high yield boost, conventional cell verification can be simple, and the ideal maintenance culture data can be obtained.

20241118

Ask:
I would like to ask the teachers, the picture is 293T cells, there are small black dots in the cells marked by the red box, and they move, what may this be, is there any treatment plan; Current attempts to treat levooxygen as mycoplasma do not seem to be effective?
It is suspected that the black spots inside the cell are some kind of intracellular microbial contamination
Get ready to test it out

Answer:
Black dot discussion: use a bottle with a dish, how long does it take to grow, the concentration of pancreatic enzyme can be lower, increase the serum concentration, replace it with a bottle imported, recover, raise it for a few days to try, if the growth time is long, the above is very effective, the product number of the serum manufacturer, the validity period of the culture medium is listed, if the time is too long, supplement glutamine, or replace it with a new batch, according to the above, if the conditions are changed to a large brand of imports, there will be a change in a few generations. If there is any effect, please write to inform us. We count black spots, slow growth problems

Black spots occur, the culture medium is not fresh, the serum nutrient content is not enough, after slow growth, the dish is easy to PH changes, resulting in greater PH changes, too acidic or too alkaline, the bottle can be improved, and can be recovered, reference. I don't think the detection is meaningful, the above should be listed, the problem can be quickly eliminated, the medium is almost the same, and the cells should be raised for a long time, and the freshness should be said to the manufacturer, or they have their own remedies. If it's wrong, please Haihan.

After verification, the problem of black spots can be eliminated quickly, and at present, several groups are verified according to the above, and users are still relatively satisfied.

Low concentration of trypsin, 0.05 is a good habit, although the digestion time is longer, the membrane is more important, several old technologies in the regular library are recommended, and do not refute the use of classmates, 0.25 We do the development of redundant reagents for automatic culture, and the concentration of trypsin is very critical.

0.25 digested cells, after passage, the cell membrane will be restored first, the cell factory is digested in a large area, we have statistics, and there are clumps of characteristic cells, 0.25 is easy to digest the outside into mud, and the inside is not digested thoroughly, and accompanied by the state of the cells old and young, especially the epithelial agglomeration, 293 is the epithelial case, the situation of the old and the young appears, the cell state is different, it is difficult to do the later experiments, reference

It is recommended to avoid the suggestion that after the formation of small clones, use a low concentration of trypsin, digest into a single passage, a large area in the digestion, old and young problems to arise, case cells, mdck

Summary: organoids, artificial meat, automatic culture, system building, microfluidic control culture and other applications, in the long run, all kinds of problems in culture must be solved or there are breakthroughs, in this regard, according to their own ability and financial resources, recommended serum-containing regimen (easy to achieve, but also serum-free culture conditions to verify a large number of cells to get the verification premise, even for clinical research application purposes, the amount of cells is also the premise of serum-free culture conditions) At present, domestic companies have launched 100 culture of organoid culture work to complete tumorigenesis and spheroidization. Expand the discussion of the following system training problem statistics (involving more statistical content, if necessary, you can leave an email)