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Method Article

Viability Assays for Cells in Culture

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DOI:

10.3791/50645

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January 20th, 2014

 ,  ,  ,  ,  ,  , 
1Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University

In This Article

Summary

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Abstract

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Introduction

The most common viability assay in the biological sciences involves cell counts. This is evidenced by an analysis of the top (most recent) 200 publications that appeared in PubMed with either of the keywords “in vitro” or “culture” on 4/29/2013 and 4/30/2013. Of these publications, 23.5% used cell count assays, including manual cell number counts, automated cell number counts with imaging software, and Trypan blue exclusion. The Live/Dead assay was used in 1% of these publications. The number of publications using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for metabolic viability was 11%. This survey of the literature also shows that the number of publications using assays such as MTT in conjunction with cell count assays was only 3.5%. Despite the trend to use one viability assay by itself, assessing cellular function in combination with cell number seems the best choice for assessing cellular integrity. Cell counts by themselves are not sufficient because the remaining cells may not be functional or healthy even though they are present in the well1,2. Conversely, functional measures such as ATP may increase or decrease in the absence of parallel changes in the number of cells. The uncoupling of metabolic readouts from cell number suggests that ATP and MTT assays should never be used as the sole viability assay. In the present report, three viability assays that survey both cellular structures and metabolic function are described, for a more comprehensive view of cellular integrity than any one assay by itself can afford.

Two of our assays require an infrared imager that measures fluorescence in the 700 and 800 nm channels. Noise is low in the infrared wavelengths, leading to higher signal-to-noise ratios3. The Odyssey imager that we use has a 4.5 log dynamic range and a bit-depth of 16, translating to 216 or 65,536 shades of infrared. This can be contrasted to 8-bit color imaging, which only affords 28 or 256 shades of color for each wavelength. Thus, 16-bit imaging has finer resolution. It should be noted that the original infrared images are often pseudocolored green (800 nm) and red (700 nm) in published reports for presentation. Odyssey imagers are commonly used both for Western blotting and In-Cell Westerns4-7. In-Cell Westerns on formaldehyde-fixed cells use primary antibodies against any protein of interest and label them in turn with infrared fluorescent secondary antibodies. This technique is known to be particularly useful for phosphorylation endpoints6. In our In-Cell Westerns, we stain fixed cells for cytoskeletal proteins α-tubulin or the neuronal microtubule associated protein 2 (MAP2) in the 800 nm channel. These proteins are abundant enough to yield high signal-to-noise ratios. We also stain our plates in the 700 nm channel for nuclei with the DRAQ5 stain and for the cytoplasm with the Sapphire stain. Both the cytoskeletal proteins and the DRAQ5 + Sapphire stains thus reflect cellular structures.

The third viability assay measures metabolic function and is called “Cell Titer Glo.” In this luciferase-based assay, luminescence values are in direct proportion to ATP levels. ATP assays are commonly used to quantify viable cells8-12. However, including the word “titer” in the name of the assay is somewhat of a misnomer because ATP output per cell can change as a function of toxin treatments and is therefore not always in proportion to cell number8. ATP levels can also be affected by circadian rhythms13 and by cell division14 and cell differentiation15. Nevertheless, the ATP assay shown here is simple to perform and useful because ATP is a robust measure of metabolic viability16-21, if not cell number per se. Using this assay to complement the infrared In-Cell Westerns therefore yields a more comprehensive picture of cellular integrity than any one assay alone.

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Protocol

A schematic of the protocols is illustrated in Figure 1.

1. Cell Plating

Plate cells in 96-well plates at different plating densities (Figure 2). For linearity checks on the N2a neuroblastoma cell line, plate 2.5k, 5k, 10k, and 15k cells per well in 3 or 6 wells/group. For linearity checks in rat primary cortical neurons, plate 25k, 50k, 100k, and 200k cells per well in 3 or 6 wells/group. If the cell lines or primary cells of interest look healthy at different plating densities, plate at and around the optimal cell density for that cell type.

Note: In the present study, N2a cells were plated in 100 μl media and primary cortical neurons in 200 μl media on plates that are designed for lower evaporation. For detailed information on cell handling, media, sera, antibiotics, and toxin treatments, please see Unnithan et al. for N2a cells8 and Posimo et al. for primary cortex cultures22.

    1. Repeat the plating on a second 96-well plate. One plate will be assayed for ATP (Figure 2A) and the other with the infrared assays (Figure 2B). One cannot use the same plate for the ATP and infrared assays because the cells must be lysed open for intracellular ATP measurements.
    2. Be sure to plate at least 3 extra wells for background subtraction in the infrared assays at the optimal plating density (column 2; Figure 2B).
  2. Add media without cells or, more inexpensively, sterile water to the outer wells in rows A and H and in columns 1 and 12. Avoid relying on data from wells along the edges, as viability is often lower here from effects of media evaporation and temperature gradients. Such problems with microplates are commonly called edge effects23,24. Do not keep these wells empty because then rows B and G and columns 2 and 11 suffer from the edge effect.
    Edge effects may be reduced by incubating a freshly seeded plate at room temperature in ambient conditions before transfer into the CO2 incubator24. Furthermore, a recent study by Carralot describes a mathematical correction method for microtiter plates using a single control column23. Oliver and colleagues used a specially designed forced air microtitration plate incubator to reduce thermal gradients25. If the edge effect is of significant concern, microplate stability chambers (e.g. BT&C Incorporated) can be purchased to create a homogenous microenvironment with high humidity and even temperature gradients.
  3. If more wells than shown in Figure 1 are needed because additional reagent dilutions or more plating densities will be tested, use the edge wells for background subtraction.
  4. Wait overnight for attachment of cells and assay the next morning as described below.

2. Luminescent ATP Assay

  1. Follow the Cell Titer Glo manufacturer's recommendations for reconstitution of the substrate with buffer and for incubation times.
    1. Remove 50 or 150 μl media from the 100 or 200 μl of plating media, respectively. Slightly less than 50 μl will remain behind in the well. Add 25 μl of the reagents (substrate plus buffer) to columns 2-6 (Figure 2A) in a 1:2 dilution.
      Note: Varying volumes of media left behind after removal could dilute or concentrate the ATP assay reagents differentially across wells. Take care to ensure that the level of liquid in all the multichannel pipette tips is equivalent. Measure the remaining liquid in select wells across the plate with a pipette immediately before adding the ATP assay reagents to ensure accuracy. If high variability exists from differential rates of media evaporation across the surface of the plate, remove all the old media and add the same volume of fresh media or phosphate buffered saline (PBS) to all wells immediately before addition of ATP assay reagents. If the plates suffer from variable levels of evaporation, try switching to the low evaporation plates from Costar Corning. In the latter plates, the 60 interior wells shown in Figure 2 only suffer from an average of 0.995%±0.41 media evaporation overnight. There is thus negligible variability in media volume at the time of assay.
    2. In columns 7 through 11, rows B through D, remove enough media to leave 50 μl behind, as detailed above, and add 50 μl of reagents in a 1:1 dilution.
    3. In columns 7 through 11, rows E through G, leave cells in 100 μl of media and add 100 μl of reagents, again in a 1:1 dilution. The company recommends that 100 μl of reagents should be diluted 1:1 in 100 μl of media.
      Note: In order to save on cost, it is possible to cut these recommendations both in volume and in dilution. However, before doing this, ensure that it does not reduce the linearity and sensitivity of the assay.
    4. Once one specific volume and dilution factor of the reagents are found to be satisfactory, stick with them in all subsequent experiments. The criteria for satisfactory data are described in the Results section.
    5. Unused reconstituted reagents can be refrozen and used at a later date to save on cost. According to the manufacturer, reconstituted reagents can be stored at -20 °C for 21 weeks with ~3% loss of activity.
  2. Place the plate on an orbital shaker for 2 min or a nutating shaker for 10 min. If part of the plate is being assayed for a different measurement and it cannot be shaken, agitate the media with simple up-and-down pipetting only in the wells of interest. However, excessive bubbles generated during this step will interfere with luminescent output.
    1. 10 min after addition of the reagents, transfer 60 μl of the well contents into white 96-well plates. Luminescence values are higher in white plates than in clear or black plates because they reflect light upwards towards the detector.
      Note: In our experience, cells survive better on the low evaporation, clear Costar plates than other plates. These specific plates are not sold with white walls. Second, the opaque-walled and clear-bottomed plates are more expensive than completely clear plates. If one transfers the luminescent liquid to white plates, the white plates can be washed and reused, saving on the cost of using new white plates for every experiment. Transferring liquid is thus the cheaper option in the long run.
    2. Pop any air bubbles prior to reading the plate with a needle or, preferably, with forced air from a plastic transfer pipette bulb. Read the plate on a luminometer with a 1 sec integration time (the company recommends 0.25-1 sec as sufficient integration time). Read the plate 10-12 min after addition of ATP assay reagents. Timing is critical for comparison across different plates, because the luminescent signal is transient with a fast decay rate, as shown by Gilbert and colleagues26.
  3. Average the luminescent values from the 3 or 6 wells in each group and plot as a function of cell number in a scatterplot (see Figure 3). First subtract average background luminescence values of the appropriate empty wells (wells 2B - 2G, wells 11B - 11D, and wells 11E - 11G) from each corresponding data point. There will be three different groups for each plating density in this initial experiment (Figure 2A). ATP levels may be linearly correlated with cell densities in one or all of these groups. Proceed with the highest dilution of reagents that still gives satisfactory results to save on cost. Criteria for satisfactory results are described in the Results section.
    Note: With the media used in the present study, background values with this assay are not high and it is possible to skip the blank wells. However, the manufacturer Promega reports differences in luminescence with different culture media. The present study uses Hyclone Fetal Clone III, a synthetic version of fetal bovine serum for N2a cells and bovine calf serum for primary cortical cultures. According to the manufacturer, calf serum decreases luminescence values but does not decrease sensitivity of the assay. Nevertheless, if this is of significant concern, dilute ATP assay reagents in PBS instead of media at the time of assay.
    1. If the cells appear to grow unevenly across columns overnight, try assaying them within 6 hr of plating. However, bear in mind that the density at the usual time of assay (for example, 24 hr after treatment) is the density at which linearity must be achieved.

3. Infrared Assays

  1. The morning after plating, fix the cells in the second plate at room temperature in a fume hood. Be sure to wear gloves for this step and dispose of the fixative as chemical waste because formaldehyde is a carcinogen. Add 4% formaldehyde and 4% sucrose in 0.1 M phosphate buffer to the existing media in a 1:1 dilution. The media can also be washed off with PBS before fixing in full-strength fixative. Either technique works well.
    1. Incubate in fixative for 20 min and then remove the fixative and wash 3x in 200 μl PBS.
  2. Store the plate in PBS with 0.2% sodium azide at 4 °C if the assay will not take place on the same day. Otherwise, proceed with the assay and place the cells in blocking solution to minimize nonspecific binding of antibodies.
    1. Dilute the fish serum Odyssey block 1:1 in PBS and add 0.3 % Triton-X 100 as a cell permeabilizer. Make enough blocking solution as well as primary and secondary antibody solutions so that 35 μl can be pipetted into each well. However, if a lot of bubbles are observed during pipetting, make >50 μl for each well, otherwise the bubbles reach down into the cells and block antibodies from binding. Excessive soapy bubbles will appear as an unstained circular spot in the middle of the well. Try to adjust the pipetting if this occurs.
    2. Incubate in this blocking solution for 30-60 min at room temperature.
      Note: 5% bovine serum albumin or normal sera from the same species as the secondary antibody can also be used as blocking solutions to save on cost of the fish serum block. Blocking solutions can affect performance of the antibody. Thus, the optimal block for each antibody is best determined empirically.
      Note: Some investigators place In-Cell Western plates on shakers during incubations. However, this is not necessary.
  3. Make the primary antibodies: 1:10,000 dilution for anti-α-tubulin (mouse monoclonal, Table 1) and 1:2,000 dilution for anti-MAP2 (mouse monoclonal, Table 1). These antibodies are specific to the proteins of interest in these cellular models; specificity is essential for any immunocytochemical stain.
    Note: MAP2 is a marker for neurons and is appropriate for mixed neuronal/glial cultures. The postnatal cultures shown here also contain some glia (~25%) because astrocytes appear first on embryonic day 18, with their numbers peaking in the early neonatal period27,28. One cannot distinguish between astrocytes and neurons in the ATP and DRAQ5 + Sapphire assays. In order to measure neurons and astrocytes separately, use simultaneous MAP2 and GFAP staining in the 800 and 700 nm channels, as described by Mullett and colleagues4,29, leaving out DRAQ5 + Sapphire. However, if all cells in the culture wish to be assessed, use a pan-cellular marker such as α-tubulin, β-actin, or GAPDH.
    Note: These dilutions have been optimized for the N2a and primary cortical cells and may not generalize to every model. Therefore, try at least two primary antibody dilutions, one on the left half of the plate and one on the right half (see Figure 2B). Alternatively, try 3-4 dilutions of primary antibodies on 3 wells each. For example, the recommended dilution on the antibody insert sheet can be flanked with two-fold changes. In other words, if the recommended dilution for immunocytochemistry is 1:500, also try 1:250 and 1:1,000 dilution factors.
    1. Dilute antibodies in 1:1 Odyssey block:PBS and add 0.3% Triton-X. To save on cost, try making the antibodies in the blocking solution that was applied to the cells in step 3.2.1 by removing this solution at the end of the incubation. Keep the cells in PBS while doing this; they must not dry out.
    2. Incubate in primary antibodies either 1-2 hr at room temperature or overnight at 4 °C. For weakly binding antibodies and proteins that are not abundant, overnight incubations can help increase signal.
      Note: Leave at least 3 wells in blocking solution for background subtraction at each secondary antibody concentration (wells 2B-2D and wells 2E-2G in Figure 2B). Do not expose these wells to any primary antibody whatsoever. They reveal the extent of nonspecific binding by the secondary antibody and are useful for calculations of signal-to-noise ratios. If there is a concern that the secondary antibodies will lead to high levels of nonspecific binding, also include control wells that are not exposed to either primary or secondary antibody but receive the same number of washes. The difference between these wells and the "secondary only" wells will reveal the extent of nonspecific binding caused by the secondary antibody alone. In our test on mouse N2a cells, signal intensity with anti-mouse secondary antibody alone was 0.557±0.032, signal with anti-rabbit secondary antibody alone was 0.533±0.041, signal with no secondary antibody was 0.357±0.003, and signal with primary and secondary antibodies was 11.867±0.911. We have thus not observed high levels of nonspecific binding even when using anti-mouse secondary antibodies on mouse cells. There are several manufacturer's for infrared secondary antibodies; we recommend only buying the highly cross-adsorbed ones. Note that the concentration of secondary IgG antibodies may vary depending upon the source.
  4. Wash off primary antibodies with 3 washes of 200 μl PBS per well, 10 min each. Primary antibodies can be saved at 4 °C for a few weeks in 0.2% sodium azide and reused until tiny specks of debris become apparent when the solutions are held up to light. This is only done to save on cost. If cost is not an issue, make fresh antibodies for each use.
  5. Dilute the secondary antibody by 1:1,000 or 1:2,000 in 1:1 Odyssey block:PBS with 0.3% Triton-X (Figure 2B). Add the 1:1,000 dilution to the top half of the plate and 1:2,000 to the bottom half of the plate. These concentrations could be further reduced to save on cost, but check for linearity before committing to this.
    Note: Be sure to add the appropriate secondary antibody solution to the background subtraction wells (column 2 in Figure 2B).
    1. If a second protein will be assayed in place of DRAQ5 + Sapphire, label cells for α-tubulin or MAP2 with 700 nm goat anti-mouse IgG and the second protein in the 800 nm channel with primary antibodies from a species other than mouse. The 800 nm channel has less background than the 700 nm channel and should be reserved for the most critical protein of interest.
    2. Incubate in secondary antibodies for 1 hr at room temperature in a drawer away from light.
  6. Wash off secondary antibodies with 3 washes of 200 μl PBS per well, 10 min each.
  7. Make the DRAQ5 + Sapphire solutions. For the left half of the plate, dilute DRAQ5 1:10,000 (0.5 μM final concentration) and Sapphire 1:1000 in PBS with 0.3% Triton-X (columns 3 - 6; Figure 2B). For the right half of the plate, dilute DRAQ5 1:20,000 (0.25 μM) and Sapphire 1:2000 (columns 7 - 10; Figure 2B).
    Note: DRAQ5 used to be sold at a 1 mM stock instead of a 5 mM stock. Some previously published reports therefore used 1:4,000 or 1:2,000 dilutions of DRAQ5 8.
    1. To save on cost, if two plates are being assayed simultaneously, the same DRAQ5 + Sapphire solutions can be used on two plates in sequence, pipetting it off the first plate and adding it to the second. If the same solutions will be used twice in this manner, use them up within one day. Diluted DRAQ5 + Sapphire solutions cannot be saved at 4 °C or frozen for later use.
    2. Incubate in these solutions for 30 min at room temperature away from light. If time is short and the DRAQ5 + Sapphire solutions will not be reused on other plates with different secondaries, add these stains to the secondary antibody solutions in step 3.5 and incubate for 1 hr.
      Note: Do not add DRAQ5 + Sapphire solution to the background subtraction wells (column 2 in Figure 2B) as these wells should not be stained in the 700 nm channel.
  8. Wash the plate 3x with 200 μl PBS per well for 10 min each. Put 0.2% sodium azide in the final PBS wash if the plate is going to be stained with other visible-range secondary antibodies after the infrared imaging is complete.
  9. Scan the plate on an Odyssey Imager. Begin by scanning the plates at intensity 5 and 169 mm resolution. Either "medium quality" or "low quality" settings are sufficient. The company does not release detailed excitation/emission filter information. However, the emission filters are approximately 20 nm wide and are centered around 720 and 820 nm, according to the manufacturer.
    Note: It is possible to scan plates while still wet as investigators may wish to continue to stain them with other markers. However, the company suggests in its online protocol that dry plates result in less well-to-well signal spread. If you scan them both wet and then dry to compare the data, be sure to remove all the PBS from the well because remaining salts after evaporation can lead to high background fluorescence along the edges.
    1. The Odyssey Imager scans up and through the bottom of the plate. Plates from different manufacturers may demand different focus offsets because the bottom of the wells can vary in their thickness and depth in the plate. Try scanning at different focus offsets and see where the highest signal-to-noise ratio and crispest (most in focus) signal is achieved: 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm. Try also to measure the depth of the well from the bottom of a plate with a ruler to confirm the findings on the Odyssey. Remember that the plastic in the bottom of the well can add additional height to the ruler measurements. The Costar plates used in the present study demand a focus offset of 4.0 mm.
    2. Use the In-Cell Western function in the software to place the right-sized grid onto the image of the plate and export the data into Microsoft Excel. Examine the "integrated intensity" values of the same wells across different focus offsets to find the highest fluorescence values. The focus offset that yields the highest signal-to-noise ratio (signal in immunostained wells versus "no primary negative control" wells) is the one to choose hereafter.
    3. Once one focus offset is decided upon, scan again in smaller increments. For example, if the brightest signal was at 3.5 and 4.0 mm, try scanning at 3.6, 3.7, 3.8, and 3.9 mm and see if there is any further improvement in signal strength. If the focus offset is wrong, signal intensities across the 12 columns on an empty plate (when all columns should have equal signal) will appear as a U-shaped curve instead of a flat line. If this continues to be a problem with plastic plates, try glass-bottomed plates.
  10. Subtract the average of the integrated intensities in the background subtraction wells (Figure 2B; wells 2B - 2D or wells 2E - 2G) from every corresponding data point in the 700 and 800 nm channels. Then average the integrated signal intensities for each group of wells. Plot the data as a scatterplot against cell density (see Figure 3).
    1. Compare the results of the two different dilutions of primary and secondary antibodies and the results of the two different dilutions of DRAQ5 + Sapphire to settle on one dilution each for subsequent experiments. If linearity is not achieved, try scanning at a different intensity. Rescan with intensities 3 and 7 instead of 5 and reanalyze the data. If the data improve at one of those intensities but are still not satisfactory, rescan in increments around that intensity.
    2. Be careful not to analyze images where the software shows white spots in the wells; those spots signify saturated signal out of the range of the imager. Scan at a lower intensity if this occurs.
    3. If linearity is not achieved, try also to fix the cells within 6 hr of plating because they might grow or die unevenly in different columns overnight. Also try different plating densities, different scanning intensities, further optimizations of reagent dilution factors, glass-bottomed plates, DRAQ5 by itself as a nucleus-only stain, or different antibodies other than anti-α-tubulin or anti-MAP2.

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Results

The rate-limiting factor in these experiments is the infrared staining, as the ATP assay is relatively brief in duration. For the infrared assays, we anticipate that eight 96-well plates can be stained and scanned within one day by staggering two batches of four plates each (see Figure 1). This estimation assumes 20 min of fixation, 30 min of washing, 30 min of blocking, 2 hr primary antibody incubation followed by 30 min of washes, 1 hr secondary antibody incubation followed by 30 min of washes, 30 min ...

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Discussion

We have found that signal strength in all three viability assays is linear and correlated with plating density. However, not all the assays are equally sensitive to 2-fold or 1.5-fold changes in plating density. For N2a cells, the infrared assays are less sensitive than the ATP assay, particularly at lower plating densities. Although the infrared assays are less sensitive than ATP, the DRAQ5 + Sapphire assays and the α-tubulin assays are in good agreement in that they reveal the highly protective impact of N-acetyl ...

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Disclosures

None of the authors have any conflicts to disclose.

Acknowledgements

We acknowledge Juliann Jaumotte for the idea of saving on the volumes of reagents in the ATP assay. We are deeply grateful for the superb administrative support of Mary Caruso, Deb Willson, and Jackie Farrer and to the Mylan School of Pharmacy for providing financial support for these studies. Thanks are also due to the Hunkele Dreaded Diseases Foundation and the Parkinson’s and Movement Disorders Foundation for their financial support of the primary neuronal studies.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Cell Titer GloPromegaG7572Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 °C for at least 21 weeks
18% FormalinThermo-Shandon9990244Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized
SucroseSigma-AldrichS0389It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells
Odyssey BlockLI-COR927-40003This fish serum can be bought in bulk and frozen at -20 °C for long term use
Triton-X 100Sigma-Aldrich21568We store a stock solution of 10% Triton-X 100 in sterile water at 4 °C
Sodium Phosphate MonobasicFisherS468One can also buy PBS tablets or 10x PBS solutions, but they are more expensive
Sodium Phosphate DibasicFisherS373See above
Sodium Azide (250x)Ricca Chemical Company7144.8-16Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 °C until they become contaminated with debris
Mouse anti-α-tubulinSigma-AldrichT5168This antibody is expensive but can be greatly diluted and is highly specific
Mouse anti-MAP2Sigma-AldrichM9942This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)
800 nm Goat anti-mouse IgGLI-COR926-32210Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source
DRAQ5BiostatusDR50200This compound used to be sold by LI-COR at 1 mM
SapphireLI-COR928-40022
LuminometerPerkinElmerVICTOR3 1420 multilabel counter
Odyssey ImagerLI-COR9201-01
Shaker/MixerResearch Products International248555

References

  1. Leak, R. K., Liou, A. K., Zigmond, M. J. Effect of sublethal 6-hydroxydopamine on the response to subsequent oxidative stress in dopaminergic cells: evidence for preconditioning. J Neurochem. 99, 1151-1163 (2006).
  2. Ugarte, S. D., Lin, E., Klann, E., Zigmond, M. J., Perez, R. G. Effects of GDNF on 6-OHDA-induced death in a dopaminergic cell line: modulation by inhibitors of PI3 kinase. J. Neurosci. 73, 105-112 (2003).
  3. Patonay, G., Antoine, M. Near-infrared fluorogenic labels: new approach to an old problem. Anal. Chem. 63, (1991).
  4. Mullett, S. J., Hinkle, D. A. DJ-1 deficiency in astrocytes selectively enhances mitochondrial Complex I inhibitor-induced neurotoxicity. J. Neurochem. 117, 375-387 (2011).
  5. Egorina, E. M., Sovershaev, M. A., Osterud, B. In-cell Western assay: a new approach to visualize tissue factor in human monocytes. J. Thromb. Haemost. 4, 614-620 (2006).
  6. Aguilar, H. N., Zielnik, B., Tracey, C. N., Mitchell, B. F. Quantification of rapid Myosin regulatory light chain phosphorylation using high-throughput in-cell Western assays: comparison to Western immunoblots. PLoS One. 5, (2010).
  7. Jinwal, U. K., Dickey, C. A. Cell-based assays for regulators of tau biology. Methods Mol. Biol. 670, 93-108 (2011).
  8. Unnithan, A. S., Choi, H. J., Titler, A. M., Posimo, J. M., Leak, R. K. Rescue from a two hit, high-throughput model of neurodegeneration with N-acetyl cysteine. Neurochem. Int. 61, 356-368 (2012).
  9. Hoskins, C., Wang, L., Cheng, W. P., Cuschieri, A. Dilemmas in the reliable estimation of the in-vitro cell viability in magnetic nanoparticle engineering: which tests and what protocols? Nanoscale Res. Lett.. 7, 10-1186 (2012).
  10. Essner, M. D., Javed, A., Eleazer, P. D. Effect of sodium hypochlorite on human pulp cells: an in vitro study. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 112, 662-666 (2011).
  11. Sims, J. T., Plattner, R. MTT assays cannot be utilized to study the effects of STI571/Gleevec on the viability of solid tumor cell lines. Cancer Chemother. Pharmacol. 64, 629-633 (2009).
  12. Petty, R. D., Sutherland, L. A., Hunter, E. M., Cree, I. A. Comparison of MTT and ATP-based assays for the measurement of viable cell. J. Biolumin. Chemilumin. 10, 29-34 (1995).
  13. Womac, A. D., Burkeen, J. F., Neuendorff, N., Earnest, D. J., Zoran, M. J. Circadian rhythms of extracellular ATP accumulation in suprachiasmatic nucleus cells and cultured astrocytes. Eur. J. Neurosci. 30, 869-876 (2009).
  14. Ataullakhanov, F. I., Vitvitsky, V. M. What determines the intracellular ATP concentration. Biosci. Rep. 22, 501-511 (2002).
  15. Iglehart, J. D., Silver, D. P. Synthetic lethality--a new direction in cancer-drug development. New Engl. J. Med. 361, 189-191 (2009).
  16. Crouch, S. P., Kozlowski, R., Slater, K. J., Fletcher, J. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J. Immunol. Methods. 160, 81-88 (1993).
  17. Kangas, L., Gronroos, M., Nieminen, A. L. Bioluminescence of cellular ATP: a new method for evaluating cytotoxic agents in vitro. Med. Biol. 62, 338-343 (1984).
  18. Lundin, A., Hasenson, M., Persson, J., Pousette, A. Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol. 133, 27-42 (1986).
  19. Sevin, B. U., et al. Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing. Gynecol. Oncol. 31, 191-204 (1988).
  20. Maehara, Y., Anai, H., Tamada, R., Sugimachi, K. The ATP assay is more sensitive than the succinate dehydrogenase inhibition test for predicting cell viability. Eur. J. Cancer Clin. Oncol. 23, 273-276 (1987).
  21. Andreotti, P. E., et al. Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian carcinoma. Cancer Res. 55, 5276-5282 (1995).
  22. Posimo, J. M., Titler, A. M., Choi, H. J., Unnithan, A. S., Leak, R. K. Neocortex and allocortex respond differentially to cellular stress in vitro and aging in vivo. PLoS One. 8, (2013).
  23. Carralot, J. P., et al. A novel specific edge effect correction method for RNA interference screenings. Bioinformatics. 28, 261-268 (2012).
  24. Lundholt, B. K., Scudder, K. M., Pagliaro, L. A simple technique for reducing edge effect in cell-based assays. J. Biomol. Screen. 8, 566-570 (2003).
  25. Oliver, D. G., Sanders, A. H., Hogg, R. D., Hellman, J. W. Thermal gradients in microtitration plates. Effects on enzyme-linked immunoassay. J. Immunol. Methods. 42, 195-201 (1981).
  26. Gilbert, D. F., et al. A novel multiplex cell viability assay for high-throughput RNAi screening. PLoS One. 6, (2011).
  27. Bayer, S. A., Altman, J. Neocortical Development. , Raven Press. (1991).
  28. Miller, F. D., Gauthier, A. S. Timing is everything: making neurons versus glia in the developing cortex. Neuron. 54, 357-369 (2007).
  29. Mullett, S. J., Hinkle, D. A. DJ-1 knock-down in astrocytes impairs astrocyte-mediated neuroprotection against rotenone. Neurobiol. Dis. 33, 28-36 (2009).
  30. Jiang, Y., et al. N-Acetyl cysteine blunts proteotoxicity in a heat shock protein-dependent manner. Neuroscience. 255, 19-32 (1016).
  31. Madeira, A., et al. Caveolin-1 interacts with alpha-synuclein and mediates toxic actions of cellular alpha-synuclein overexpression. Neurochem. Int. 59, 280-289 (2011).
  32. Fioriti, L., et al. Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations. J. Biol. Chem. 280, 11320-11328 (2005).
  33. Zhang, L., et al. Proteasome inhibition modulates kinase activation in neural cells: relevance to ubiquitination, ribosomes, and survival. J. Neurosci. Res. 87, 3231-3238 (2009).
  34. Braak, H., et al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol. Aging. 24, 197-211 (2003).
  35. Stranahan, A. M., Mattson, M. P. Selective Vulnerability of Neurons in Layer II of the Entorhinal Cortex during Aging and Alzheimer's Disease. Neural Plast. 2010, (2010).
  36. Duyckaerts, C., Delatour, B., Potier, M. C. Classification and basic pathology of Alzheimer disease. Acta Neuropathol. 118, 5-36 (2009).
  37. Chu, C. C., Tranel, D., Damasio, A. R., Van Hoesen, G. W. The autonomic-related cortex: pathology in Alzheimer's disease. Cereb. Cortex. 7, 86-95 (1997).
  38. Braak, H., Del Tredici, K., Bohl, J., Bratzke, H., Braak, E. Pathological changes in the parahippocampal region in select non-Alzheimer's dementias. Ann. N.Y. Acad. Sci. 911, 221-239 (2000).
  39. Braak, H., Rub, U., Schultz, C., Del Tredici, K. Vulnerability of cortical neurons to Alzheimer's and Parkinson's diseases. J. Alzheimers Dis. 9, 35-44 (2006).
  40. Calabrese, E. J. Hormesis is central to toxicology, pharmacology and risk assessment. Hum. Exp. Toxicol. 29, 249-261 (2010).
  41. Giordano, J., Ives, J. A., Jonas, W. B. Hormetic responses in neural systems: consideration, contexts, and caveats. Crit. Rev. Toxicol. 38, 623-627 (2008).
  42. Mattson, M. P. Hormesis defined. Ageing Res. Rev.. 7, 1-7 (2008).
  43. Wang, P., Henning, S. M., Heber, D. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS One. 5, (2010).
  44. Riss, T. L., Moravec, R. A. Use of multiple assay endpoints to investigate the effects of incubation time, dose of toxin, and plating density in cell-based cytotoxicity assays. Assay Drug Dev. Technol. 2, 51-62 (2004).
  45. Titler, A. M., Posimo, J. M., Leak, R. K. Astrocyte plasticity revealed by adaptations to severe proteotoxic stress. Cell Tissue Res. , (2013).
  46. McLaughlin, B., et al. Caspase 3 activation is essential for neuroprotection in preconditioning. Proc. Natl. Acad. Sci. U.S.A. 100, 715-720 (2003).
  47. Mathews, S. T., Plaisance, E. P., Kim, T. Imaging systems for westerns: chemiluminescence vs. infrared detection. Methods Mol. Biol. 536, 499-513 (2009).
  48. Picariello, L., et al. A comparison of methods for the analysis of low abundance proteins in desmoid tumor cells. Anal. Biochem. 354, 205-212 (2006).
  49. Tapias, V., Cannon, J. R., Greenamyre, J. T. Melatonin treatment potentiates neurodegeneration in a rat rotenone Parkinson's disease model. J. Neurosci. Res. 88, 420-427 (2010).
  50. Fenteany, G., Schreiber, S. L. Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells. Chem. Biol. 3, 905-912 (1996).
  51. Omura, S., et al. Lactacystin, a novel microbial metabolite, induces neuritogenesis of neuroblastoma cells. J. Antibiot. 44, 113-116 (1991).

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