Cell culture is the complex process by which cells are grown under controlled conditions, generally outside of their natural environment.

• SENTHIL KUMAR Sankareswaran

  Does anyone have photos of mycoplasma contaminated cell lines (especially HepG2, A549 or MCF7) ?

  We are facing a lot of contamination problems.

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  • 
    SENTHIL KUMAR Sankareswaran replied

    Is it possible to see in microscopy?

    1 minute ago
• Indhumathi Veerappan

  How to identify mycoplasma contamination?

  I am able to identify fungal contamination easily by the finger like hyphae formation. But am confused with bacterial and mycoplasma contamination, while I also want to know about chemicalI am able to identify fungal contamination easily by the finger like hyphae formation. But am confused with bacterial and mycoplasma contamination, while I also want to know about chemical contamination analysis.

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    Indhumathi Veerappan replied

    thanks all.. we have A549, Mcf-7 cell lines in our lab. recently we encounter a problem with cells getting detached after 2 or 3 days in gud condition.. so we doubt it might be due to contamination.thanks all.. we have A549, Mcf-7 cell lines in our lab. recently we encounter a problem with cells getting detached after 2 or 3 days in gud condition.. so we doubt it might be due to contamination. can anyone comment on problem in using HEPES in media? I read dat increased exposure of HEPES containing media to fluorescent light might cause damage to cells

    8 hours ago
• mj mm

  HeLa suspension cells - Spinner culture

  I am looking for a detailed cell culture protocol for spinner cell culture of HeLa cells.

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    A N Singh Rajput replied

    HeLa SPINNER CULTURES induced by Interferon alpha and gamma: 4 c detail vist..HeLa SPINNER CULTURES induced by Interferon alpha and gamma: 4 c detail vist.. "http://www.sigmaaldrich.com/etc/medialib/docs/Sigma-Aldrich/Instructions/1/ecacc_handbook.Par.0001.File.tmp/ecacc_handbook.pdf" "http://tools.invitrogen.com/content/sfs/manuals/des_man.pdf" and " http://genome.ucsc.edu/ENCODE/protocols/cell/human/HeLa-S3_IFN_Crawford_protocol.pdf"

    1 day ago
• Neeraj K Satija

  Cells detaching in culture?

  I am facing a problem with all cell lines and primary cells I am using like hMSCs, SaOS2, HEK, NIH3T3. Since past about a week, the cells have started to detach from the surface and this detachmentI am facing a problem with all cell lines and primary cells I am using like hMSCs, SaOS2, HEK, NIH3T3. Since past about a week, the cells have started to detach from the surface and this detachment is more rapid in petridishes and plates. In culture flasks also, the cells are detaching from the neck side and gradually progressing down the flask. I borrowed medium and a different cell line from another lab, and those cells also detached once kept in our incubator. I checked the incubator temperature with thermometer and it is OK. Also the pH of all media I am using is fine. Any suggestions please what is happening with the cultures?

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    Elena S Nadezhdina replied

    Check the quality of CO2, sometimes it contains CO.

    2 days ago
• Indhumathi Veerappan

  Debris inside the cells?

  Cells during initial passages appear clear, but after few lines of passages black dots like debris are seen inside the cells. Are these normal or occuring due to contamination? The cells seem to beCells during initial passages appear clear, but after few lines of passages black dots like debris are seen inside the cells. Are these normal or occuring due to contamination? The cells seem to be normal and are attached.

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    Brajesh Varshney replied

    black specs are a nemesis to a cell culturist. I have seen it in CHO cells and tried to understand their implications in long time culture. The scale up of such culture would almost always pose ablack specs are a nemesis to a cell culturist. I have seen it in CHO cells and tried to understand their implications in long time culture. The scale up of such culture would almost always pose a problem eventually getting contaminated. I might agree with Hossein that they are contaminants and the best way is to start with a new culture

    2 days ago
• Hyo-Yeon Kim

  The shape of J774A.1 cell line

  Hello, This picture is J774A.1 cell line. I wonder whether the cells on the right side of picture, and middle (upper) of picture are stressed. They are cultured in DMEM with 10% FBS and 1%Hello, This picture is J774A.1 cell line. I wonder whether the cells on the right side of picture, and middle (upper) of picture are stressed. They are cultured in DMEM with 10% FBS and 1% antibiotics. I only have a experience in culturing RAW264.7 cell line, and I have not seen the cell showing that shape in normal condition. Although you can see a very small number of unusual cell, there are cells as many as round-shaped cells in the culture flask, actually. The cells are purchased from ATCC and passaged twice. Thank you. Sincerely, Hyo-Yeon Kim

  
• Ismail Elshimy

  Does anyone have a detailed protocol for the isolation of microglial cells from mouse brain using percoll?

  Microglia isolation using percoll

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    Lucia Whitman replied

    Here is a protocol for you to try. There is a reference at the bottom of the protocol that should give more information.

    Flow Cytometry - Resident Glia (MPH).docx

    3 days ago
• Anna Muellner

  Tamoxifen stability in medium

  I need to induce my cells using 4-OHT. Does anyone know how long it remains active in normal DMEM medium at 37°C 5%CO2?

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    Frank Eertmans replied

    Previously,I have been using tam in comparable conditions for several days (5-7 days) without any problem. You can also refresh medium with tam every 48h but normally seeb, it should be no problem.Previously,I have been using tam in comparable conditions for several days (5-7 days) without any problem. You can also refresh medium with tam every 48h but normally seeb, it should be no problem. Good luck!

    3 days ago
• junaid Ahmed Kori

  How to avoid contamination from my media and plates?

  I have often done plating and inoculation and all that, but right now I am facing a problem of the contamination in my media even when it is growing in unstreaked plates. I have changed the platesI have often done plating and inoculation and all that, but right now I am facing a problem of the contamination in my media even when it is growing in unstreaked plates. I have changed the plates and bought new ones, cleaned the autoclave machine, autoclave media properly, changed the deionized distilled water too, sterilized the incubator with ethanol, sterilized laminar hood every time at the time of inoculation (with UV sterilization for 30 minutes and ethanol sterilization). Could anybody suggest where my mistake is.

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    junaid Ahmed Kori replied

    Well thanks to all for sharing such kind of meaningful information to me.

    4 days ago
• Elisabeth Stein

  Does anybody know a method to isolate M-cells?

  My topic is innate immunity of mucosal surfaces, I am especially interested in epithelial M cells. Does anybody know a protocol to isolate them?

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    Ajay Krishnamurthy replied

    PLease check this article............ Microbial Pattern Recognition Receptors Mediate M-Cell Uptake of a Gram-Negative Bacterium INFECTION AND IMMUNITY, Jan. 2006, p. 625–631 Vol. 74, No. 1

    4 days ago
• Giulia Bena'

  Calcium phosphate transfection: does anyone successfully use it for transient experiments?

  I found several protocols online and tried one for stable transfections in HeLas. It seems to work but 90% of my cells died, the remaining were very sick. Also I noticed that some protocols say toI found several protocols online and tried one for stable transfections in HeLas. It seems to work but 90% of my cells died, the remaining were very sick. Also I noticed that some protocols say to use very small amount of DNA of interest and to equalize the DNA content using Bluescript plasmid DNA; others instead use massive amount of DNA of interest without Bluescript. Any suggestions?

  http://www.flemingtonlab.com/Protocols/CalciumPhosphateTransf.pdf

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    yu bo replied

    What you said also happened to me. I think the CaPO4 is not suitable for Hela cells. So does Lipo2000. You can try some reagent from Roche company.

    6 days ago
• Shambhavi Shubham

  Cells detaching from the plate after reaching confluency.

  Hello Everyone, I am working with fetal bovine aortic endothelial cells and I treat them with specific growth factors eventually. Usually they are very elongated and have protusions when dividing .Hello Everyone, I am working with fetal bovine aortic endothelial cells and I treat them with specific growth factors eventually. Usually they are very elongated and have protusions when dividing . WHen I seeded the cells on Thursday they looked fine but on Sunday they looked a little bit thinner and not healthy and had started to come off the plate. Still I went ahead with addition of only medium without serum. Could anyone please guide me as to what could be the reason for cells sloughing off. Also, I was wondering whether cells when become confluent they enter G0 phase so would they still respond to growth factors ? Any suggestions would be really appreciated. Thanks, Shambhavi

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    Shambhavi Shubham replied

    Thanks all for your suggestion. The cells have almost detached and don't look healthy so probably I will have to postpone my growth factor treatments. I would make sure from next time that I keepThanks all for your suggestion. The cells have almost detached and don't look healthy so probably I will have to postpone my growth factor treatments. I would make sure from next time that I keep changing my media every 2 days to keep them healthy. Thanks again for all the suggestions.

    6 days ago
• Imran Khan

  I am making M9 minimal medium but seeing no cell growth, where am I going wrong?

  My medium composition is Na2HPO4 0.6% KH2PO4 0.3% NaCl 2% NH4Cl 0.1% CaCl2 1mM MgSO4 0.2% I am autoclaving all medium contents together except CaCl2(filter sterilized) and MgSO4 (filterMy medium composition is Na2HPO4 0.6% KH2PO4 0.3% NaCl 2% NH4Cl 0.1% CaCl2 1mM MgSO4 0.2% I am autoclaving all medium contents together except CaCl2(filter sterilized) and MgSO4 (filter sterilized), I would be really happy if anyone can provide their thoughts or suggestion on this. I am using LB medium for preculture, and when I inoculate into the minimal medium, I used to give wash with distilled water. But still my organism (Vibrio) unable to grow in this minimal medium.

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    Golla Ramanjaneyulu replied

    Hi Imran Basically for the growth of any organism Carbon and nitrogen source is impartant for their metabolism, i think without Carbon source there is no growth,add any carbon source as minimum asHi Imran Basically for the growth of any organism Carbon and nitrogen source is impartant for their metabolism, i think without Carbon source there is no growth,add any carbon source as minimum as you can and then observe.

    7 days ago
• Barbara Capuccini

  Adehesion to the substrate of BV2 cells

  I haven't a good adeshion of Bv2 cells in the 6 wells plates using polylysine solution. Anyone knows a protocol that works?

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    Barbara Capuccini replied

    I'll try, thank you

    10 days ago
• Cunhua Shao

  How to culture bone-derived MSCs?

  I have been trying to culture bone marrow mesenchymal stem cells of rat for months, but the cells trended to ageing and change from fusiform shape to wide and flat in P1. Cells are cultured withI have been trying to culture bone marrow mesenchymal stem cells of rat for months, but the cells trended to ageing and change from fusiform shape to wide and flat in P1. Cells are cultured with DMEM/F12 supplemented with 10%FBS(Gibco, 16000-044), 1 mM Sodium Pyruvate , 50 uM 2-Mercaptoethanol and antibiotic-antimycotic. Can anyone advise what I should do now? Should i add bFGF and EGF ?

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    Mahmood Saba Choudhery replied

    Hi, we use this DMEM or IMDM supplemented with 15 - 20% FBS +Streptomycine and penecillin additionally you can add sodium pyruvate, non-essential amino acids i hope this will help

    10 days ago