Description
This In-Cell ELISA (ICE) Support Pack is for use with suspension, apoptotic/detaching cells and adherent cell lines. The pack contains five 96-well microplates, buffers and protocol to perform ICE with MitoSciences ICE-validated antibodies. For an ICE assay, it is necessary to purchase both primary antibody(ies) and labeled secondary antibody(ies). Antibodies are sold separately, allowing customizing the target(s) of interest, method of detection and multiplexing.
Principle
In-Cell ELISA uses quantitative immunocytochemistry to measure protein levels or post-translational modifications within cells. The cells are fixed to the bottom of a coated 96-well plate (provided). Targets of interest are detected by primary antibodies, which are in turn quantified with labeled secondary antibody(ies). MitoSciences offers highly-specific, well-characterized primary antibodies, IRDye®- or HRP-labeled anti-mouse and anti-rabbit secondary antibodies, as well as IRDye®-labeled isotype specific anti-mouse antibodies. By combining antibodies of different species or isotype and appropriate IR-labeled secondary antibodies, two color multiplexing can be achieved in the 800/700 channels. IR imaging and quantification is performed using a LI-COR® Odyssey® or Aerius® system. HRP-labeled complexes are developed and quantified colorimerically using spectrophotometer.
Two protocols are available when using this kit. Protocol (1) suspension cells and cells likely to detach under experimental conditions (for example, adherent cells undergoing apoptosis readily detach from a culture plate). Protocol (2) - A second protocol is available for normal adherent cells. These protocols can be used with any of MitoSciences' ICE-validated antibodies. Specific scientific information, background and working concentration for each antibody are detailed in each antibody's corresponding Technical Data Sheet.
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Figure 1
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Figure 1. Suspension cells are efficiently crosslinked to the assay plate. Untreated (CON) or 4 hour Staurosporine-treated (STS, 1 µM) Jurkat cells were seeded at 200,000 cells per well into the assay plate in media containing 10% bovine fetal serum (10F Medium) or in PBS, and fixed as described in the MS922 Protocol. After the fixation the number of cells attached (fixed) to the plate was determined and expressed as percentage of total seeded cells. Mean and standard error of the mean (n=2) is shown. Note that virtually all cells (untreated or STS-treated) attached to the plate whether the fixative was added to cells in 10F media or PBS.
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Figure 2
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Figure 2. Suspension cells remain firmly attached to the assay plate within the ICE assay. Untreated (CON) or 4 hour Staurosporine-treated (STS, 1 µM) HL-60 cells were seeded in the indicated amounts into the assay plate in media containing 10% bovine fetal serum (10F Medium), media without serum (0F Medium) or PBS, and fixed as described in the MS922 Protocol. The cell amount attached to the assay plate was determined by Janus green staining either just after the fixation (Panel A) or at the end of ICE assay (Panel B). Mean and standard error of the mean (n=2) is shown. Note: (1) virtually all fixed cells remained attached to the plate within the duration of the ICE assay (compare the cell amounts in Panel A to the cell amounts in Panel B), (2) that the STS-treated cells attached nearly as efficiently as the untreated cells and (3) that the cells attach efficiently even in media containing 10% serum.
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Figure 3
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Figure 3. Apoptotic adherent and suspension cells are efficiently crosslinked to the assay plate. Adherent cell lines were seeded (HeLa and HepG2 at 50,000 per well, H196 at 150,000 per well) directly in assay plate, allowed to attach overnight and treated with 1 µM Staurosporine (STS) as indicated. Suspension cells were treated with 1 µM STS or 50 ng/mL Fas antibody as indicated, concentrated by centrifugation, re-suspended in media containing 10% serum and transferred (HL-60 at 300,000 per well, Jurkat at 200,000 per well) to the assay plate. Cells were fixed as described in the MS922 Protocol and the plate was analyzed by ICE to measure the cleaved PARP using MSA43. Mean and standard error of the mean (n=3) is shown. (A) Relative levels of apoptosis measured as PARP cleavage normalized to cell amount measured by Janus Green whole cell stain. Note HepG2 cells are resistant to undergoing apoptosis under these conditions, consistent with all of our previous observations. (B) Cell amounts measured by Janus Green. Note no or very small differences between Janus Green staining of treated and untreated cells indicating that the treated cells undergoing apoptosis are efficiently crosslinked to the assay plate.
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Figure 4
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Figure 4. Apoptotic suspension cells are efficiently crosslinked to the assay plate. HL-60 cells were seeded as indicated into a 96-well plate and treated with 1 µM Staurosporine or vehicle for 4 hours. The treated cells were directly transferred to the 96-well assay plate. Cells were fixed as described in the MS922 Protocol and the plate was analyzed by ICE to measure the cleaved PARP using MSA43. (A) Cell amounts measured by Janus Green. Mean and standard error of the mean (n=6, coefficient of variation 0.05 or less) is shown. Note no or very small differences between Janus Green staining between vehicle-and Staurosporine-treated cells indicating that the Staurosporine-treated cells undergoing apoptosis are efficiently crosslinked to the assay plate. (B) Relative levels of apoptosis measured as PARP cleavage normalized to cell amount measured by Janus Green whole cell stain. Mean and standard error of the mean (n=3) for cells seeded at 300,000 per well is shown.
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