Hey all,
Im having a hard time figuring out the most optimal way of preparing a cell lysate for my situation. I hope you can help.
So here is the case:
Im culturing IL-1α & IL-1β Reporter HEK 293 Cells (https://www.invivogen.com/hek-blue-il1r) which are modified in such a way that it can colorimetrically quantify nfkappa beta pathway activity when relevant cytokines are present in the culture medium (such as IL1a/b, Il1RN, mouse TNF-alpha etc).
Now I would like to deterimine the nfkappa beta activating potential of my tumor cell lines (of which i additionallt take along the conditioned medium of these cell lines). In order to do this, i want to add cell lysate of these cell lines on top of the cultured HEK cells (20uL is suggested according to the manual) in 96 well plate format. Obviously id rather not use lysis buffer prepared cell line protein. At the same time i would like to have a pure protein preparation without too much debris and dna.
Would really appreciate if you guys have any suggestions
thank you very much for your consideration and time
Why don't you coculture the cells?. At the end of the day, only the ones (HEK293) carrying the detector (SEAP) will light up. As a control, use a regular HEK293 clone.
hi oscar, thanks for your answer. It did cross my mind to indeed just perform a co-culture but because i have large variability between proliferation rate of my tumor cells i was thinkink it might skew my readout too much. Nonetheless, its a good solution I will have to try out. Starting with an x-number of tumor cells for each tumor cell line in my co-culture, you just have to accept the inherent nature of growth rate and cytokine production in that setup.
Hi Marnix H. P. de Groot . It's just an idea. The first experiment though, is making a calibration curve, trying to answer the question how many HEK293 cells you need to get a detectable signal. One scenario is to plate your HEK293 cells at 2 densities (25% and 50% confluency). Next day or whenever cells look healthy and subconfluent, treat cells with a full dose range of the corresponding cytokine but focusing on the low level of detection (for IL-1beta reporter cell line, treat with IL-1b). For an additional negative control, get a parental HEK293 cell line as well (no SEAP, not a reporter cell line). - In both cell lines (reporter and parental) detect Alkaline Phosphatase. It's important to run these controls because one of your cancer cells might express TNAP (Tissue Non-specific Alkaline Phosphatase). So there are 2 negative controls: from the parental cell line (endogenous expression of TNAP) and the other negative control (reporter cell line, SEAP with zero transcriptional stimulation). Let's say that the 50% confluence of HEK293 gave you a detectable signal at low IL-1b concentrations. COCULTURE: Then, you're set because in your experiment you can plate the cancer cell line with the reporter cell line in the same well (coculture). It's very easy to do, but it's critical to rule out the possibility that endogenous Alkaline Phosphatase might be high and not useful to quantitate cytokine production by cancer cell lines. In that case, it's best to get Luciferase reporter cell lines.
Dear Marnix H. P. de Groot , you may proceed with the Bead beating protocol to make lysis which will allow to make lysate the whole cell without use of chemical. Silica beads of certain size are used to beat the cells but you need a bead beater (vortexer) to do so. at the end of the protocol you will find the pure lysate.
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