Principle of the Assay

NF-κB is typically present and resides in the cytoplasm of most cells as a complex with members of IκB inhibitor family of proteins. Both the size of this complex and IκB masking of the nuclear localization sequence of NF-κB prevents NF-κB entry through the nuclear membrane. Once IκB is phosphorylated and degrades, the nuclear localization sequences become assessable and NF-κB is free to translocate to the nucleus (Figure 3). Microscopy images reveal strong evidence of the redistribution of NF-κB from the cytoplasm to the nucleus following cytokine stimulation (Figure 4).

Figure 3:

Diagrammatic view of the principle of NF-κB activation. NF-κB (green), normally resides in the cytoplasm, once activated by stimuli (IL-1α, TNF-α ,etc) it translocates to the nucleus.

Figure 4:

HeLa cells unstimulated or stimulated with 25 ηg/ml IL-1α for ~40 minutes. Cells were then fixed and labeled with NF-κB-p65 polycloncal antibody and secondary AlexaFluor488 with counterstain of Hoechst 33342. (Left). Unstimulated (more...)

Image Analysis Algorithm to Detect NF-κB Translocation

The basic principle to detect NF-κB translocation is dependent on identifying the nucleus of a cell using one of many nucleic acid probe reporters, typically Hoechst, DAPI, or DRAQ5.

Once the nucleus is identified using image analysis process it is masked, and then a secondary mask overlay is created to either encompass the entire cell boundaries or a subset area of the cytoplasmic area. Within this secondary masked area (nucleus and/or cytoplasm) labeled NF-κB can then be quantified. Most available algorithms report two critical features; (A) NF-κB intensity in the nucleus and (B) NF-κB intensity in the cytoplasm. Once determined, a translocation value is calculated by measuring the average intensity of “difference” of the NF-κB protein between the identified cytoplasmic region and nuclear region (Cyto-Nuc Difference). Additionally, a “ratio” of the average intensity of the nuclear region to average intensity of the cytoplasmic region can be calculated to represent this translocation value; Nuc/Cyt Ratio. Both the “difference” and “ratio” features reflect a biological phenotype of NF-κB translocation. The data feature output from images is commonly reported as a well summary value but the calculation begins at the single cell level as shown in Figure 5. An example of well level data nomenclature describing these key features from different imaging platforms is showcased in Table 1. Representative images and algorithm overlays of HeLa cells in a dose response with IL-1α are shown in Figure 6. Differences at the plate level (96 wells) in cells treated with stimuli (Max) or with DMSO or inhibitor compound (Min) are used to calculate Z-factor values (34) using CytoNuc Difference and Nuc/Cyt Ratio, highlighted in Table 2.

Figure 5:

Multiparametric plot (Top) and corresponding images (Bottom) of NF-κB-AF488 average cytoplasm ring and nucleus circ intensities at the single cell level in HeLa cells following TNF-α dosing for 35 minutes. Each “dot” or (more...)

Table 1:

Algorithm nomenclature output comparison of HCS feature data to measure NF-κB autofluorescence only.

Figure 6:

Nuclear Factor κB Translocation in HeLa cells following 30 minute incubation with IL-1α. Cells labeled with Hoechst 33342 are masked with a blue ring outline to identify the nuclear area. Dilation of this mask to identify cytoplasm area (more...)

Table 2:

NF-κB-p65-AF488 HCS feature data output comparison of Nuc/Cyt Ratio and CytoNuc Difference measurements in HeLa cells. The minimum (min) signal is the baseline constitutive expression of NF-κB and the maximum (max) is NF-κB response (more...)

Additional, there are other valuable HCS output features that are useful including identifiable objects (cells), variability of intensity across area within the nucleus or cytoplasm, morphological measurements including the size, shape, length, aspects of individual cells or objects within a cell. For compound screening, the use of nucleus or cytoplasm area intensity may also be helpful to identify nuisance fluorescent compounds that display the same or similar spectral properties as the probes used in the assay design and development. However, keep in mind that these compounds could also be classified as “false positive” activate compounds from the screen. In this case, careful follow ups needs to be employed b measuring basal autofluorescence levels in cells without fluorescent reporters and with compound(s) in question.

Assay Detection and Limitation

The assay described below will quantify the redistribution of NF-κB from the cytoplasm to the nucleus upon activation using antibodies to NF-κB p65 subunit in intact cells following fixation. Stimulating cells with proinflammatory cytokines such as IL-1α and TNF-α activates the canonical NF-κB pathway and therefore the noncanoncial pathway (CD40, LT), or the atypical IκK independent pathway (UV, Hypoxia) will likely be non-responsive in this assay format. In addition, this assay can indirectly measure IκB degradation since phosphorylation of IκB is required before degradation of IκBα and subsequent translocation of NF-κB subunit to the nucleus. This assay does not directly measure phosphorlyation of NF-κB protein.

This assay can be multiplexed with other biofluorescent probes. For example, other kinase related bioprobes to measure MAPK kinase pathways such as phospho-JNK-1/2 or viability indicators may provide important information about the biology or selectivity of the compound. The critical consideration for developing this assay is the dose and time using more than one stimulus to triggering multiple signaling pathways.

Cell Model

HeLa cells are endothelial cells from the American Tissue Culture Corporation (ATCC), catalogue number CCL-2, original isolated from the cervix of a 31yo black female with adenocarcinoma, were selected as the cell line as choice for this assay for several reasons. (A) HeLa cells are well documented in the literature to illustrate NF-κB redistribution; (B) HeLa cells are relatively flat once seeded in plates and therefore image well using widefield 2-D bright-field and fluorescent microscopy; (C) there are commercial available validated kits and antibodies to detect redistribution of NF-κB.

Note: Other cells such as Swiss 3T3, BHK, HepG2-C3A, Rat hepatocytes, LLCPK, HMVEC, CHO-K1, HT1080, HCT-116, U 2-OS, and others are also known to express NF-κB but before adopting one of these cell lines or other unlisted cell models, it is recommended to validate assay performance with a known cell line such as HeLa.

Cell culturing and harvesting

Maintenance of the HeLa cell line or cell of choice should be carried out following recommendations from the ATCC or the source of the cell line. Typically splitting cells 2-3 times per week is required for optimal growth and performance in the assay. Do not allow cells to become over confluent in tissue culture flasks. If this occurs, it will be necessary to thaw a new vial of cells before proceeding with assay development, validation, or screening. Grow cells in complete media, and then adjust serum concentration prior to cell seeding as necessary (see Effects of Serum for additional information). Upon harvest of cells with trypsin or other cell detachment methodology it is recommended to filter the cells through a cell strainer (40-70 microns) to remove large clumps before cell counting and seeding. If aggregates are not removed this will negatively impact cell object identification and segmentation with image analysis algorithms.

Cell Seeding Number Determination

Determine the optimal cell seeding density as described in the assay development HCS chapter by seeding cells at approximately 5,000 cells/well for a 96-well plate as the median starting point; then dilute or increase the cell density by approximately 40-50%.(Note: In 384-well plates, approximately 2,500 cells/well is recommended). Incubate cells overnight, and then label as described in subsequent sections with reporter probes. There is some variability but no significant difference in cell number (3,000 to 10,000 cells/well) in 96-well plate format from the NF-κB inhibitor compound IC₅₀ curves (Figure 7). However, since identification and segmentation of individual cells in the well is optimal for accurate image analysis measurement of NF-κB, 5,000 cells/well was chosen. An NF-κB reference compound inhibitor, IK202, was used to determine cell number. The number of cells per well was optimized based on reviewing images to observe cell-to-cell contact. Higher concentrations of cells per well resulted in rejection of more cells due to piling of cells, massive cell contact clumps, and poor image analysis segmentation. However, the number of fields required to collect 500 cells, the assay defined threshold, was decreased (Figure 8). The optimal cell seeding density for HeLa cells is 5,000 cells/well for 96-well plate. {data not shown}).

Figure 7:

Cell Number Determination. HeLa cells seeded overnight, treated with reference compound inhibitor for 15 minutes and subsequent 25ηg/ml TNF-α for ~35 minutes, and then fixed and stained for NF-κB-p65-AF488. Plates were analyzed (more...)

Figure 8:

Field numbers required to collect 500 objects (cells) using a 10X/0.3NA objective, no magnifier and a 1392x1024 camera pixel array sensor.

Cell Passage Limitations

It is critical to gauge the number of cell passages in the assay before a noticeable decline is observed. Cells with many passages may not survive, become contaminated, or fail to respond in the assay over time. If possible, continue passaging cells at the onset of development with a fresh thaw of cells and maintain a weekly stock over time; then measure NF-κB response in comparison with lower passage cells. HeLa cells with different cell splitting passage numbers up to 40 from ATCC and the same lot were tested to compare sensitivity to TNF-α cytokine to activate translocation of NF-κB and to determine if the response was altered. Even thought the R2 of the curve fitting was similar; there was a significant shift in the NF-κB translocation EC50 response to cytokines in cells with high passage number. A significant loss in responsiveness of NF-κB translocation was observed with an increase in calculated EC₅₀ values directly correlated with increasing passage number (Figure 9). Based on these findings it is recommended to use cells with as low passage number as possible and as a general rule never exceed passage of 20.

Figure 9:

Cell Passage Number Comparison. Different cell splitting passages of HeLa cells seeded at 5,000 cells/well overnight and treated with dose response of TNF-α for ~35 minutes, then fixed and stained to measure NF-κB translocation. Plates (more...)

Activators of the NF-κB Signaling Pathway

Activating NF-κB pathway using cellular cytokines as described in the introduction is critical to establish an assay window. Although several stimuli have been identified in activating NF-κB pathway, three cytokines, IL-1α, IL-1β, and TNF-α gave the most robust and best EC₅₀ values of all stimuli tested (Figure 10). IL-1α, IL-1β family members and TNF-α cytokines activate NF-κB through different up-stream mechanisms. Platelet-derived growth factor (PDGF) also activates the translocation events of the NF-κB pathway to a lesser extent and is described to work through Ras and PI3 kinase signaling. (33). Lymphotoxin (LT) α1/β2, LT α2/β1, and IFN-γ showed little or no activation of NF-κB (the latter stimuli data is not shown). Additionally, anisomycin and phorbol-12-myristate-13-acetate (PMA) were also tested but did not show NF-κB-p65 activation as robustly as IL-1α, IL-1β, or TNF-α stimulus. This knowledge is important if considering multiplexing NF-κB with other signaling pathways. In this assay design, development, and validation, HeLa cells will be stimulated with both IL-1α and TNF-α following compound treatment, however, for compound profiling and screening, either stimuli alone will work and choice of cytokine is depended on the upstream or downstream target and biology.

Figure 10:

Activation of NF-κB-p65 with Different Stimuli. HeLa cells seeded at 5,000 cells/well overnight and treated with stimuli for 30 minutes, then fixed and labeled with NF-κB-p65-AF488 and Hoechst33342 to measure NF-κB translocation. (more...)

Plate Type

The NF-κB assay is considered a very robust HCS assay using HeLa cells and likely can be adapted to many different SLAS standard plate types including 384-well or even 1536-well formats, however it is recommended to first perform the proof of concept in a validated format such as the 96-well, as discussed within, then move to another well plate format. In this assay sterile 96-well tissue cultured treated Perkin Elmer (Packard) View plates were chosen with no extracellular matrix proteins or PDL substrate coating.

Reagent & Probes

There are a few validated assay kits and antibodies in market that can be used for this assay, or alternatively development of an in-house or “homebrew” assay protocol kit can be developed. In the next section, details of developing and comparing with a commercially available kit are described. The primary advantage of using a commercial kit is it is “ready to use” with “cookbook” instructions included. If just measuring a few plates, then this is a preferable option. The benefit of developing an in-house “homebrew” kit is to know what antibodies, buffers, and reagents are being used in the assay as this may not be disclosed by commercial sources of kits. In addition, the cost for running many plates is reduced once an in-house kit is developed. This chapter should reduce development time and costs. An example comparing a commercial kit with an in-house kit using alternative antibody sources and the process to cross validate their relative performance is described in next section.

Note: Before beginning the assay development process determine if a fixed endpoint assay is appropriate for the biological question. Alternatives to the classic antibody recognition and binding approach is the use of a fusion protein of NF-κB with a fluorescent protein reporter such as GFP, mCherry, or HaloTag® (Promega, Madison, WI) are other options to consider for measuring protein function.

Commercial Kit Validation & Development of In-house “Home-Brew” Kit

The initial assay development was performed using commercial available validated kits for NF-κB. Additional, several different commercial available antibodies were screened for signal-to-noise ratio performance that targeted the p65 subunit of NF-κB (Appendix-1). Although other antibodies worked well in the evaluation, based on performance and comparable EC₅₀ calculated values, the rabbit polyclonal IgG NF-κB-p65 antibody from Santa Cruz (SC-372) was used for assay development and validation procedures (see Figure 11).

Figure 11:

Comparison of Commercial and HomeBrew Buffers. HeLa cells seeded at 5,000 cells/well overnight and treated with dose response of IL-1α for ~35 minutes, then fixed and stained to measure NF-κB translocation using SC-372 antibody. Plates (more...)

Since the commercial kit contains other unknown and perhaps proprietary reagents, i.e., antibody, buffers, and reagents including PBS; PBS containing 0.5% Triton X-100; PBS containing 0.01% Tween-20 for testing and comparison with “in-house” buffers and commercial kit; it is always important to verify the signal-to-noise ratio window and insure that assay variability is not sacrificed .

Comparisons of different lots of NF-κB antibody from Santa Cruz were done at three different concentrations of primary antibody following stimulation with IL-1α. There were no significant differences observed (Figure 12). Lot-to-lot variability was evaluated several times with new antibody lot shipments and no significant differences in the calculated EC₅₀ values were observed.

Figure 12:

Lot-to-Lot Variability Comparison of Santa Cruz SC-372 NF-κB-p65 Antibody. HeLa cells seeded at 5,000 cells/well overnight and treated with dose response of IL-1α for ~35 minutes, then fixed and stained to measure NF-κB translocation (more...)

Fixation comparison of two different forms of formaldehyde solution

Formaldehyde, 37% stock solution (Sigma) and Ultra-pure Formaldehyde, methanol-free 10% stock solution (Polysciences) were compared. HeLa cells were treated with IL-1α for about 30 minutes, followed by 1:10 dilution of fixation buffer in PBS for 10 minutes. An approximately 3-fold shift in the EC₅₀ values was observed (Figure 13). For this assay, 3.7% formaldehyde from Sigma containing methanol was used. It is possible higher concentrations of Ultrapure formaldehyde may be used but this needs confirmation by testing.

Figure 13:

Comparison of Formaldehyde Fixatives. HeLa cells seeded at 5,000 cells/well overnight and treated with either dose response of IL-1α (left) or TNF-α (right) for ~35 minutes, then fixed with either 3.7% formaldehyde or 1% Ultrapure Formaldehyde (more...)

Note: At the time of testing 16-20% paraformaldehyde solution was not readily available from commercial sources and should be considered as alternative source.

Please refer to institutional safety guideliens before working with formaldehyde; suggestions are in Safety Consideration Guidelines Precautions. Formaldehyde is specifically regulated by OSHA, so be sure you are in compliance with the OSHA standard (http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=10075&p_table=STANDARDS).

Stability and Lot Variability of Cytokines

Variability of cytokines lots were measured to determine variability of the assay if new reagents were introduced to the assay during the screening campaign or used in subsequent screens. Please note, it is highly recommended to order all reagents before the start of the experiment and/or screen. From this experiment and several other experiments, both IL-1α (data not shown) and TNF-α EC₅₀ responses and calculated EC₅₀ values indicated very high reproducibility (Figure 14). In addition, the activity and stability of cytokine performance was measured after one or more freeze-thaw cycles (Figure 15).

Figure 14:

Lot variability of tumor necrosis factor alpha. HeLa cells seeded at 5,000 cells/well overnight and treated with dose response of TNF-α for ~35 minutes, then fixed and stained to measure NF-κB translocation. Plates were analyzed on HCS (more...)

Figure 15:

Stability of cytokines following multiple freeze-thaw cycles. Following the reconstitution of the cytokine per manufacture suggestion, cytokine reagents were store at –80^oC, and then allowed to thaw at room temperature before use. Samples were (more...)

Stability of NF-κB detection in cells following fixation

The NF-κB protein in HeLa cells is stable for several days at 4°C prior to staining with NF-κB antibody following fixation with 3.7% formaldehyde for 10 minutes, washed and stored in PBS. This provides flexibility in screening operations if a “Stop Point” is required in the workflow. It is recommended that cells stained with NF-κB antibody be imaged as soon as possible, and not to exceed 14-days (Figure 16). For logisitics in screening operations including robotics, liquid handling, and cell plate handling to reduce flutations in pH and temperature, 35 minutes was choosen.

Figure 16:

Stability of NF-kB-p65-AF488 complex post staining and fixation. Using several plates, HeLa cells seeded at 5,000 cells/well overnight and treated with 25ηg/ml of TNF-α for ~35 minutes, then fixed and stained to measure NF-κB translocation (more...)

Time Course of Cytokine Stimulation

Determine the optimal time window for NF-κB translocation by performing a time dependent stimulation with known stimuli. HeLa cells seeded at 5,000 cells/well in a 96-well plate overnight were then treated with 50 ηg/ml of IL-1α to establish a time course for activation and redistribution of NF-κB. Following incubation with cytokine, cells were fixed at different times and then labeled with NF-κB antibody using an indirect staining method with secondary fluorescent antibody. Images of cells expressing fluorescent antibody are captured using HCS imager and analyzed for NF-κB expression. Time ½ = 24 minutes (Figure 17). Based on these results 30-60 minutes proved to be an excellent window to capture NF-κB translocation.

Figure 17:

NF-κB Translocation time course kinetics. HeLa cells seeded at 5,000 cells/well overnight and treated with 25ηg/ml of IL-1α over time, at 5 or 10 time minute intervals, cells were fixed and then stained to measure NF-κB (more...)

Effects of Serum

HeLa cells treated with cytokines IL-1α and TNF-α were tested with different concentrations of fetal bovine serum (FBS) to determine if serum affects the assay performance and window. While the assay window did not diminish with concentration of serum, the sensitivity of cytokine stimulation as calculated by EC₅₀ values of NF-κB translocation was significantly different; IL-1α sensitivity with 0.5% FBS increased ~3-fold as compared to 10%FBS, while TNF-α sensitivity increased by ~ 2.5-fold using 0.5%FBS as compared to 10%FBS (see Figure 18). Based on this data and subsequent data 0.5% FBS concentration was chosen for validation experiments.

Figure 18:

NF-κB translocation effects from serum concentrations. HeLa cells seeded at 5,000 cells/well overnight in 10% Fetal Bovine Serum (FBS), were then removed from complete serum by washing with serum-free media and replaced with 0.5%, 1%, 2%, or 10% (more...)

DMSO Tolerance

HeLa cells seeded at 5,000 cells/well in 100µL media containing 0.5%FBS were allowed to attach overnight. Cells were then incubated with DMSO concentrations up to 10% for 30 minutes (2X the dosing time for compound treatment) followed by 30-minute incubation of cytokine. The increase and subsequent decrease in signal at DMSO concentrations greater than 1% on the graph is a result in change in cell morphology and cytotoxicity respectively (Figure 19). At 5% DMSO, the cell’s cytoplasm area shrunk as a result of increase in nuclear size, thus the image analysis algorithm measurement is reflective in the data showing an increase in CytoNuc translocation (see figure 19). At 10% DMSO, the cells are fragmented, dissociated, or detached from the plate and therefore not possible to quantify cytoplasm or nuclear fluorescent expression.

Figure 19:

DMSO tolerance. HeLa cells seeded at 5,000 cells/well overnight and treated DMSO for 15 minutes followed by stimulation with either media only containing 0.5% FBS, 25ηg/ml IL-1α, or 25ηg/ml TNF-α for ~35 minutes. Cells (more...)

Dosing Time of compounds on Cells

It is highly recommended to pre-determine the duration of exposure dose of an inhibitor compound prior to stimulation with secondary stimuli such as cytokines or growth factors. Please keep in mind and consider the following:

• Is it possible to co-add the inhibitor compound and stimuli simultaneously?
• How does this affect the S:N window or assay variability?
• Is a pre-incubation required to maximize window and assay variability?
• Is there an advantage for the workflow in screening operations?

For this assay a 15 minute pre-incubation of inhibitor compound was chosen to make certain the compound exposure to the cells was saturated prior to stimulating with cytokines. Comparative data suggests simultaneous co-addition of both the inhibitor compound and stimuli does not affect the performance of the assay window (data not shown). But keep in mind these are reference compounds and unknown chemicals in a compound library may not readily penetrate cells before NF-κB is activated.

Inhibitors of NF-κB Pathway

There are several known inhibitors of NF-κB pathway that have been published such as Bayer’s compound, BAY 11-7085, which was initially selected as the control inhibitor reference compound for assay development and validation because it was commercially available and showed IC₅₀ activity of about 10 μM (Figure 20). Additionally there were two compounds identified in-house named IK101 and IK202 with improved potency. These compounds were chosen as reference compounds in the assay validation, although BAY 11-7085 and BAY 11-7082 compounds both could serve as reference compounds. The IK101 and IK202 compounds, with IC₅₀ values of less than 2 μM and 0.3 μM, respectively, were used for demonstration and performance for assay validation purposes (Figure 21). Inhibitor compounds were pre-incubated with cells for 15 minutes followed by cytokine addition for about 30 minutes.

Figure 20:

BAY 11-7085 structure and NF-κB translocation inhibition experimental variability. From 3 experiments, HeLa cells seeded at 5,000 cells/well overnight and treated in dose response with reference compound inhibitor, BAY11-7085 for 15 minutes followed (more...)

Figure 21:

Comparison of NF-κB translocation inhibition using reference compound inhibitor compounds BAY 11-7085, IK101 and IK202. HeLa cells seeded at 5,000 cells/well overnight and treated in dose response with reference compound inhibitors, BAY11-7085, (more...)

Reference Compound Stability of Freeze-Thaw Cycles

HeLa cells treated with reference compounds (IK101 or IK202) that underwent multiple freeze-thaw cycles over a course of several weeks were tested for stability and/or loss of activity. Compounds were dissolved in 100% DMSO and aliquoted in cryovials and stored at –80°C for later use. After up to 10 freeze-thaw cycles there is no significant evidence of loss of compound activity (Figure 22).

Figure 22:

Freeze-thaw cycle stability of reference compound inhibitors IK101 and IK202. Reference inhibitor compounds were made at 10mM in DMSO and stored at –80^oC, and then allowed to thaw at room temperature before use. Compound vial samples were then (more...)

Performance and Validation Pre-Screen

Following the optimization in the prior steps it is critical to validate the process procedure for screening operations before testing unknown chemicals. In Figure 23 and Figure 24, the reproducibility of cytokine stimuli is determined over a course of three independent assays and at least 3 different days to mimic the time it may take to screen an entire library. This requires independent harvesting of cells from flasks, cell seeding, and so on over 3 or more days. In Figure 25, the reference compound inhibitor IK202 was tested for reproducibility over 3 independent experiments, again to mimic screening operations. The results were acceptable for proceeding with Z-factor determination.

Figure 23:

IL-1α dose response experimental variability. HeLa cells seeded at 5,000 cells/well overnight and treated with IL-1α in dose response for ~35 minutes were fixed and then stained to measure NF-κB translocation. Plates were analyzed (more...)

Figure 24:

TNF-α dose response experimental variability. HeLa cells seeded at 5,000 cells/well overnight and treated with TNF-α in dose response for ~35 minutes were fixed and then stained to measure NF-κB translocation. Plates were analyzed (more...)

Figure 25:

Reference compound inhibitor IK202 dose response 3 day experimental variability. HeLa cells seeded at 5,000 cells/well overnight and treated in dose response with reference compound inhibitors IK202 for 15 minutes followed by stimulation with 25ηg/ml (more...)

To determine the robustness and variability of single dose addition across the entire plate using liquid handling and robotics, at least 2 full plates with minimum and maximum responses are required to calculate a Z-factor. Z-factor is calculate using the formula 1- [3*(standard deviation of positive control + standard deviation of negative control) / (mean of positive control – mean of negative control)] (33). The values from the Z-factor are indicative of the variability of the data, values of less than 0 is considered too much overlap in the positive and negative signal; 0 – 0.5 is considered a marginal assay and 0.5 – 1 is considered an excellent assay.

For the NF-κB assay, the maximum response (Max or positive) is cytokine addition and the minimum (Min or negative) response is the reference compound inhibitor, i.e., IK202 + cytokine. Additional a Mid response can be added to verify ~50% reduction in the assay window. As mentioned earlier and throughout this chapter measuring translocation of NF-κB with HCS can use one or more HCS data features, namely intensities of the Cytoplasm-Nucleus Difference (CytoNuc Diff) or the Nucleus to Cytoplasm Ratio (Nuc/Cyt Ratio). In Figure 26 (IL-1α) and Figure 27 (TNF-α), both the CytoNuc Diff and Nuc/Cyt Ratio is reported. Interestingly, the TNF-α stimuli showed equivalent Z-factor values, however, the IL-1α stimuli Z-factor was significantly different; the CytoNuc Diff was 0.76 and the Nuc/Cyt Ratio was 0.64. Both are considered excellent screenable assays.

Figure 26:

NF-κB translocation IL-1α stimuli Z-factor calculation & graph display. HeLa cells seeded at 5,000 cells/well overnight into 3 different 96-well plates were treated with 0.5% DMSO for maximum response (Red), media for unstimulated (more...)

Figure 27:

NF-κB translocation TNF-α stimuli Z-factor calculation & graph display. HeLa cells seeded at 5,000 cells/well overnight into 3 different 96-well plates were treated with 0.5% DMSO for maximum response (Red), media for unstimulated (more...)

Reagents for assay are listed in Table 3.

Table 3:

List of materials and reagents

Preparation of Stock Reagents:

1.

Reference compound (IK202), 10 mM: Make a 10mM stock solution by dissolving 10mg IK202 compound (MW=202.3) into 4.95 ml DMSO. Store at –80°C.

2.

Hoechst 33342, 2 mg/ml: Make a 2 mg/ml stock solution by dissolving in 100% DMSO. Protect from light. Store aliquots at -80°C ~indefinitely.

3.

IL-1α, 10 µg/ml: Make a 10 μg/ml stock solution by dissolving in PBS containing 0.1% BSA. Store aliquots at -80°C for up to 6 months.

4.

TNF-α, 10 µg/ml: Make a 10 μg/ml stock solution by dissolving in PBS containing 0.1% BSA. Store aliquots at -80°C for up to 6 months.

Working Solutions per 96-well Plate (Prepared daily)

1.

Inhibitor compound: Dilute in EMEM media without serum at 5X final concentration (50 μM final at 250 μM). For IK202 compound, dilute 25 μl of 10mM stock into 1ml of EMEM media. Note final DMSO concentration should not exceed 1%.

2.

IL-1α: Make working solution at 6X (150ηg/ml) the final assay concentration, which is 25ηg/ml. Dilute 15 μl of 10 μg/ml cytokine into every 1ml of EMEM media needed to complete the assay. For one 96-well plate dilute 45 μl of 10 μg/ml cytokine stock in 3ml of EMEM. Note, for EC₅₀, start at 40ηg/ml final and dilute 1:3 in PBS or medium. For time course or kinetics study, use 25 ηg/ml.

3.

TNF-α: Make working solution at 6X (150ηg/ml) the final assay concentration, which is 25ηg/ml. Dilute 15 μl of 10 μg/ml cytokine into every 1ml of EMEM media needed to complete the assay. For one 96-well plate dilute 45 μl of 10 μg/ml cytokine stock in 3ml of EMEM. Note, for EC₅₀, start at 40 ηg/ml final and dilute 1:3 in PBS or medium. For time course or kinetics study, use 25 ηg/ml.

4.

Formaldehyde: Dilute 37% formaldehyde 1:10 with PBS. Warm to 37°C before use, this must be prepared fresh with each assay run.

5.

0.1% Triton X-100: Dilute 250 µl of 10% Triton X-100 in 24.75 ml of PBS. Prepare fresh daily.

6.

0.01% Tween-20: Dilute 50 μl of 10% Tween-20 in 50ml of PBS. Prepare fresh daily.

7.

Primary Rabbit anti-NF-κB-p65 polyclonal antibody: Make 5 μg/ml 1^o antibody solution by diluting antibody 1:40 in PBS (137.5 μl of antibody into 5.5ml of PBS). Prepare fresh daily.

8.

Goat anti-Rabbit Alexa-488 Secondary Antibody / Hoechst Stain: Make a 10 μg/ml 2^o antibody solution by diluting antibody 1:400 in PBS (27.5 μl of 2^o antibody into 5.5ml of PBS). Add 5.5 μl of 2 mg/ml Hoechst 33342 stock solution for a 2 μg/ml solution (1:1000 dilution). Prepare fresh daily and protect from light.

Protocol for finding Inhibitors (Antagonist) in the NF-κB Pathway

Note: All washes and buffers are at 100μl unless indicated otherwise.Assays can be performed with automated hand-held pipettes for less than 10-plates. For more than 10-plates it is recommended to use automated plate washers and liquid handling devices.

Day-1

1.

Seed approximately 5,000 cells/well in 100 μl volume of EMEM media containing 0.5% FBS, 2 mM L-Glutamine in 96-well plate (recommend using automated cell dispensing device for uniformity)

2.

Allow cells to attach overnight in complete medium at 37°C, 5% CO₂, 95% relative humidity.

Day-2

3. Transfer 25 μl 37°C pre-warmed compound from v-bottom compound plates to cell plate using automated liquid handling device.

4. Incubate for 15 minutes at 37°C, 5% CO₂, 95% relative humidity.

5. Transfer 25 μl 37°C pre-warmed cytokine (IL-1α or TNF-α) from v-bottom compound plates to cell plate using automated liquid handling device.

6. Incubate for 35 minutes at 37°C, 5% CO₂, 95% relative humidity.

7. Remove all media using hand-held aspirator or with automated liquid handling device.

Note: option to add high concentration of fixative directly to the cells or remove media and add final concentration of fixative buffer.

8. Immediately fix cells by adding 100 μl of “pre-warmed” 3.7% formaldehyde solution in a vented hood or with an automated liquid handling device.

9. Incubate at room temperature for 10 minutes with plate lid.

10. Remove formaldehyde and replace with 100 μl of PBS using an automated liquid handling device. Please read safety precautions before working with formaldehyde.

Note: At this step, you can stop the experiment and store plate at 4^oC for several days by filling wells with salt solution and sealing to prevent evaporation.

11. Add 100 μl of 0.1% Triton X-100/PBS working solution and incubate 5 minutes at room temperature (RT). Use automated liquid handling device as needed.

12. Wash plate 2X with 100 μl PBS at RT, leaving PBS on cells.

13. Use 96-head aspirator to remove buffer or use an automated liquid handling device. DO NOT ALLOW WELLS TO DRY.

14. Immediately add 50 μl of 1^o antibody using hand-held pipettor or automated liquid handling device. Incubate for 60 min at RT.

15. Remove antibody and discard. Wash plate 1X with 100 μl of 0.01% Tween 20/PBS.

16. Incubate for 15 minutes at RT.

17. Wash 2X with 100 μl PBS at RT, leaving PBS on cells.

18. Use 96-head aspirator or automated liquid handling device to remove buffer. DO NOT ALLOW WELLS TO DRY.

19. Immediately add 50 μl of 2^o antibody containing Hoechst dye per well.

20. Incubate 1-hour in the dark at RT. Protect from light by using black tape plate lids or foil.

21. Wash with 100 μl of 0.01% Tween20/PBS. Incubate for 10 minutes at RT.

22. Wash plate with 100 μl PBS at RT

23. Add 200 μl PBS and seal plates.

24. Analyze plates on High Content Imaging device. If required, plates can be store at 4^oC in dark for future analysis. Do not freeze plates.

Compound Libraries

It is recommended to use a library of compounds to test the sensitivity and robustness of the assay for both negative and positive responses. There is a unique NF-κB inhibitor set of 14 -compounds available from Calibiochem (Cat#481487 InhibitorSelect™ NF-κB Signaling Pathway Inhibitor Panel). There are now 4 versions of this library with the latter ones containing IκK inhibitors. Additional there are other larger compound library collections commercially available for purchase including LOPAC. For this assay the Calbiochem kinase Library, otherwise known as the Millipore EMD 539744 InhibitorSelect™ 96-Well Protein Kinase Inhibitor Library-I was the only commercial library available. Interestingly, this 80 chemical compound library contains no NF-κB or IκK inhibitor compounds and this is reflected in the results. For single point determination at 50 μM, two compounds showed an increase in NF-κB CytoNuc difference was a result in changes in the morphological characteristics of the cell in assay both with IL-1α and TNF-α stimulation (Figures 28 and 29). No activates were confirmed with IL-1α or TNF-α stimulation IC₅₀ curves follow-ups from 50μM, 3-fold in duplicates.

Figure 28:

Results from Calbiochem kinase library (EMD 539744 InhibitorSelect™) screen. HeLa cells seeded at 5,000 cells/well overnight and dosed with a single dose of 50µM compound for 15 minutes followed by stimulation with 25ηg/ml TNF-α (more...)

Figure 29:

Reference compound and unknown inhibitor dose response with corresponding images. Top panel is a comparison of reference compound inhibitor and two unknown inhibitors identified as actives in screen. HeLa cells seeded at 5,000 cells/well overnight and (more...)

Other Considerations

Detection of Cytotoxicity and Fluorescent Compounds in NF-kB Translocation Assay

One of the powers of HCS is providing the capability to measure morpohological and intensities of bioprobe markers at the single cell level. The combination of these capapbilites allows the ability to measure cell populations and subpopulations captured in the image. If a compound dosing concentration is the direct or indirect cause of cytotoxicity it is often measureable with good precision by using population statistics of the number of cell objects detected in the well in comparison to a vehicle control well. Optional, cytotoxic and viable dyes can be used in combination with the biomarker indicator as an reporter. HeLa cells seeded overnight, then pre-incubated with 10-point compound dose response starting at 50µM, ½ log for 15 minutes and susequently treated with TNF-α for 35 minutes, were staiend with NFkB antibody as previously described. The inhibition of NF-κB in the presence of the known reference compound recorded an IC₅₀ of 0.2095 µM using 3-parametric fit in GraphPad Prism, whereas the unknown inhibitor compounds recored and IC₅₀ of 0.2679 µM and 0.6685 µM. The differences in these values is somewhat subtle in the curve fitting; however, the key differences is observed in the loss of cells with increasing concentration of the compound above the recorded IC₅₀ value. It is important to always review and recored the number of cellular objects, fields, or other HCI feature parameters that provide information about an alteration in the cell seeding density in the well in addition to other key HCS feature values including reviewing the images to validate the results (Figure 30).

Figure 30:

SpotFire 3-D graph visulaization: well level plate HCS features showing separation between normal cell distrubution, altered morhology, or cell loss and/or cytotoxic effects. X axis = number of valid fields; Y axis = valid object count; Z axis = average (more...)

To determine if an unknown compound(s) is fluorescent in the visible spectra when screening a chemical compound library using HCS assays can be achived by using intensity measurements in the nucleus or cytoplasm regions of interests (ROI). These compounds can create a false positive and are not commonly identified until follow ups are underway in protein redistrubution assay such as NFkB where fluorescence can occupy both the cytoplasm and nuclear ROI. If a suspect compound is thought to be fluorescent in the HCS assay, this can verify using a high content imager by measuring the baseline autofluorescence of cells using vehicle control, then measure any increase in fluorescence following compound treatment. Evidence of compound fluorescence in the cells is typically observable using fluoresent mcirscopy images not shown.

Formaldehyde and Paraformaldehyde Use Guidelines in the Laboratory:

1.

All stock solutions greater than 4% must be maintained in a fume hood.

2.

Working solution of less than 4% and less than 37°C can be used in the following areas:

• Fume hood
• Biosafety cabinet
• Automation liquid handling robots with standard setup with <4% formaldehyde solution in appropriate reservoir positions. Following addition of formaldehyde, plate lids must cover open plates to minimize formaldehyde exposure during a 10-15 minute incubation period. Transfer of formaldehyde to and from the liquid handling robot reservoir requires a safety container that is larger than the stock reservoir container to collect any spill. It is recommended that the volume of working solution of formaldehyde does not exceed 100ml at any given time. If additional formaldehyde solution volume is required than add as needed.

3.

Transport of formaldehyde working solution between laboratories and pre-warming to 37°C requires standard lid cover to prevent potential exposure.

4.

Formaldehyde waste should be less than 0.1% before it is discarded; be sure to verify per institution or government ordinance.

5.

Formaldehyde mixture with DMSO requires normal DMSO disposal procedures.

6.

Stock solutions of 37% formaldehyde contains methanol and disposal of this stock solution should be treated as flammable if appropriate.

7.

Report any excessive spill of formaldehyde to your safety department