In the world of cell culture, RAW264.7 is a special presence.<\/p>\n\n\n\n
It is a mouse macrophage cell line, and fields such as immunology, inflammation, oncology, and drug screening deal with it almost every day. But anyone who has cultured it knows \u2014This cell line has a bad temper and a mind of its own.<\/strong>With a little carelessness, it becomes \u201cpolarized\u201d: extending pseudopodia, spreading and adhering, turning into a spindle-shaped or polygonal form, and even starting to phagocytose and secrete factors.<\/p>\n\n\n\n For many experiments,the most important prerequisite is precisely<\/strong>that they do not polarize. For example, in drug regulation or gene function studies, you want to see real changes induced by the drug, not false positives caused by the cells actively differentiating on their own.<\/p>\n\n\n\n So here comes the question:How exactly should RAW264.7 be cultured so that it stays nicely round and undifferentiated?<\/strong><\/p>\n\n\n\n First, learn to read its appearance. The first lesson in culturing RAW264.7 is to understand its mood through the microscope.<\/p>\n\n\n\n What troubles many beginners the most is: they looked round just yesterday, but today after a medium change, they\u2019ve all spread out. What\u2019s the problem?In most cases, the serum is to blame.<\/strong><\/p>\n\n\n\n RAW264.7 is extremely sensitive to endotoxins, hormones, and growth factors in serum. Ordinary fetal bovine serum may contain trace amounts of LPS (lipopolysaccharide) or other stimulants, which can induce M1 polarization in RAW264.7 even at very low concentrations.<\/p>\n\n\n\n Solution<\/strong>\uff1a Many laboratories have tested our serum, and the conclusion is consistent:with the same cells, after switching to our serum, they became round.<\/strong><\/p>\n\n\n\n Recommended passage ratio<\/strong>\uff1a1:3 to 1:6, passage every 2\u20133 days. It is best to passage when the cells are maintained at 70%\u201380% confluency. Do not let the cells reach 100% confluency, otherwise extensive differentiation will occur.<\/p>\n\n\n\n RAW264.7 cells are semi-adherent. During passaging, wash once with PBS, add trypsin (0.05% with EDTA) to cover for 1\u20132 minutes, gently tap the culture dish, and observe under a microscope. When the cells become rounded but have not completely detached, immediately stop digestion by adding serum-containing culture medium.<\/p>\n\n\n\n Avoid using<\/strong>a cell scraper or vigorous pipetting \u2014 that will damage the cell membrane and induce a stress response.<\/p>\n\n\n\n RAW264.7 cells are very sensitive to stimulation by fresh serum. Frequent medium changes (e.g., every day) is like repeatedly giving them a \u201cshot of adrenaline.\u201d Generally, changing the medium every 2 days is sufficient, or change it during passaging.<\/p>\n\n\n\n In addition to serum, culture media, PBS, trypsin, and even pipette tips can introduce endotoxins. It is recommended touse endotoxin-free PBS and culture media,<\/strong>and to pay attention to aseptic details during operation.<\/p>\n\n\n\n Mycoplasma contamination does not necessarily cause the culture medium to become turbid, but it can continuously activate the immune response of RAW264.7 cells, leading to gradual differentiation. If you find that cell condition has inexplicably deteriorated or morphology has changed, it is recommended to test for mycoplasma. We provide aMycoplasma Removal Reagent<\/strong>that can prevent or eliminate contamination with low cytotoxicity.<\/p>\n\n\n\n Not all fetal bovine sera are suitable for culturing RAW264.7. We specifically use RAW264.7 as aquality control cell line,<\/strong>and every batch of serum is validated by RAW264.7 culture before release:<\/p>\n\n\n\n Many customers have reported that after switching to our serum, RAW264.7 cells finally \u201cbehave.\u201d They no longer readily spread and differentiate, the experimental background for drug stimulation is clean, and data reproducibility is greatly improved.<\/p>\n\n\n\n Of course, some experiments do require RAW264.7 cells to polarize into macrophages (for example, for phagocytosis or inflammatory cytokine release). In such cases, you can stimulate them with LPS (100 ng\/mL) or IFN\u2011\u03b3.However, note that the results are reliable only when you start stimulation from the resting, round state.<\/strong>If the cells are already partially differentiated, adding stimulation will lead to different baselines, and the data will not be comparable.<\/p>\n\n\n\n Therefore, the core strategy for culturing RAW264.7 is:keep it round and \u201casleep\u201d under normal conditions, and wake it up only when needed.<\/strong><\/p>\n\n\n\n RAW264.7 is not difficult to culture, as long as you understand it. We manufacture fetal bovine serum, and we also act as the \u201cguardian\u201d of RAW264.7. Feel free to contact us to request a trial sample of serum specifically for RAW264.7.<\/strong> \u5728\u7ec6\u80de\u57f9\u517b\u7684\u4e16\u754c\u91cc\uff0cRAW264.7 \u662f\u4e00\u4e2a\u7279\u522b\u7684 …<\/p>","protected":false},"author":1,"featured_media":27,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[1],"tags":[],"class_list":["post-1","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-uncategorized"],"_links":{"self":[{"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/posts\/1","targetHints":{"allow":["GET"]}}],"collection":[{"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/comments?post=1"}],"version-history":[{"count":5,"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/posts\/1\/revisions"}],"predecessor-version":[{"id":682,"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/posts\/1\/revisions\/682"}],"wp:featuredmedia":[{"embeddable":true,"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/media\/27"}],"wp:attachment":[{"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/media?parent=1"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/categories?post=1"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/www.celleasy.com.cn\/en\/wp-json\/wp\/v2\/tags?post=1"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n\n\n\n1. Three typical states of RAW264.7<\/h2>\n\n\n\n
Status<\/th> Morphological characteristics<\/th> What does it represent?<\/th><\/tr><\/thead> Undifferentiated (Ideal)<\/strong><\/td> Round, bright, with good refractility, in suspension or semi-adherent, easily detached by gentle pipetting.<\/td> The cells are in a resting state, which is most suitable for comparative experiments before and after stimulation.<\/td><\/tr> Partially differentiated (Caution)<\/strong><\/td> Cells extend a few pseudopodia, becoming irregular or small spindle-shaped. They can still be passaged, but their state has already changed.<\/td> Suboptimal culture conditions (serum stimulation, too low density, endotoxin, etc.), experimental results begin to become uncontrollable.<\/td><\/tr> Fully differentiated (Discard)<\/strong><\/td> Cells spread into large patches, become spindle-shaped or polygonal, adhere firmly, and are difficult to detach by pipetting.<\/td> Essentially differentiated into a macrophage-like phenotype, no longer suitable for studies requiring a resting state.<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n
\n\n\n\n2. Six key points for RAW264.7 culture<\/h2>\n\n\n\n
\uff081\uff09. Serum is the biggest variable \u2014 don\u2019t let it cause polarization.<\/h3>\n\n\n\n
Choose fetal bovine serum with extremely low endotoxin levels and stable batch-to-batch consistency.Our premium\/special grade fetal bovine serum<\/strong>is tested for endotoxin at \u22645 EU\/mL per batch (often even lower in practice) and contains no LPS-like bioactive substances. When culturing RAW264.7 with our serum,the cells maintain a round, non-polarized resting state for extended periods,<\/strong>remaining morphologically stable even after more than 10 passages.<\/p>\n\n\n\n\uff082\uff09Density control: not too dense, nor too sparse.<\/h3>\n\n\n\n
\n
\uff083\uff09 Passage method: gently pipette, do not scrape hard.<\/h3>\n\n\n\n
\uff084\uff09Medium change frequency: Don\u2019t be too diligent.<\/h3>\n\n\n\n
\uff085\uff09Avoid sources of endotoxin contamination.<\/h3>\n\n\n\n
\uff086\uff09 Mycoplasma contamination \u2013 the invisible driver of polarization<\/h3>\n\n\n\n
\n\n\n\n3. Our serum keeps RAW264.7 from \u201cgrowing up.\u201d<\/h2>\n\n\n\n
\n
\n\n\n\n4. A tip: When is polarization needed?<\/h2>\n\n\n\n
\n\n\n\nSummary:<\/h2>\n\n\n\n
It fears stimulation, endotoxins, improper density, and mycoplasma.
And what it fears most is a \u201cbad-tempered\u201d bottle of serum.<\/p>\n\n\n\n
If you are struggling with the condition of this cell line, give our serum a try \u2014
keep it round, just as it should be.<\/strong><\/p>\n\n\n\n
\n\n\n\n
(You can also use our new press-to-mix culture medium, low-endotoxin PBS, penicillin-streptomycin, and mycoplasma removal reagent as a complete system, making RAW264.7 culture simple and stable.)<\/p>","protected":false},"excerpt":{"rendered":"