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HelpCorner - xCELLigence System

General

FAQ: How can I save time when using the xCELLigence System?

HelpCorner: Automated monitoring and data collection contribute to significant time savings when using the xCELLigence System, with only a few minutes of extra setup time (Figure 1).


For example, in a 96-well plate, 32 different conditions can be tested in triplicate; the wells need to be seeded with cells, monitored, and sampled.


For both manual and xCELLigence System experiments, it takes approximately 1 hour to prepare the 32 conditions (including dilution series and mixing of components). Plating of the cells is more straightforward on the E-Plate, because a manual experiment involves manipulation of a number of vessels (e.g., 96 T25 flasks) to obtain relevant data for cell counting.


Programming the xCELLigence System takes 10-20 minutes. Adding cell medium to the E-Plate and performing an initial background measurement takes another few minutes.


Once the xCELLigence System begins recording, everything is automatically monitored online; by comparison, a manual experiment generally requires about an hour per day to obtain qualitative information on the cultures. In addition, a manual experiment that uses cell counting requires significant additional work for each of the 96 wells, including removal of medium, washing, trypsinization, collection, centrifugation, and resuspension (~5 minutes per well, plus a few minutes per sample for counting).


Screening with the xCELLigence System is about 7 times faster than manual procedures for a simple experiment; this can increase to tenfold in more complicated manual experiments in which medium changes and extra components are involved. Time savings are even more pronounced when the xCELLigence System is compared with manual experiments in which multiple time points are required to generate data (such as doubling time).


FAQ: In the G-Protein Coupled Receptor (GPCR) Appli–cation Note 6, following excitation with agonist, the peak subsides and then drops below the initial Cell Index and sometimes drops down to negative values [1]. What does this mean? Are these doses toxic to the cells, resulting in cell death or detachment?


And, does a sharper peak indicate a quick termination of signal, and a flatter peak a longer return to baseline?


In addition, does the “tail end” of the curve reflect anything? For example, if after stimulation the Cell Index is higher in some cells than others, is this biologically significant?

HelpCorner: The shape of the curve is a reflection of the signaling pathways being modulated within a given cell type. In order to correctly interpret a particular GPCR signature profile, many parameters need to be taken into consideration, including the signaling pathway being activated, the dose of the agonist/antagonist, the receptor density, and the confluency of the cells.


GPCR responses are primarily a reflection of the inherent morphological and adhesive changes that take place within the cell. At times, these transient changes in morphology/adhesion can lead to negative Cell Index readings. For certain types of receptors, the response can be very quick, while for others it can take much longer.


FAQ: How is the CIM-Plate 16 organized?

HelpCorner: The CIM-Plate 16 is composed of an upper chamber (UC) and a lower chamber (LC). The underside of the UC is sealed by a porous polyethylene terephthalate (PET) membrane. The underside of the PET membrane features an array of gold microelectrode sensors. The UC and LC snap together to form a perfectly sealed device. Chemoattractant is added to the wells of the LC and cells are added to the wells of the UC.

Applications


FAQ: Is it possible to transfect cells directly in the E-Plate wells?

HelpCorner: Yes, you can perform your ­transfection directly in the E-Plate wells. Consider using direct or reverse transfection procedures, both of which have been successfully used in E-Plate wells. For experimental details, please consult Cell Analysis Application Notes 3 and 4 [2, 3].


FAQ: How can I apply different doses of radiation when using the xCELLigence System?

HelpCorner: There are several different ways by which various doses of radiation can be applied:

a. The cells are seeded in a few wells (e.g., a row or ­column), allowed to attach and grow, then irradiated with a given dose. The irradiated cells are then seeded to different wells of the same plate, allowed to attach and grow, then irradiated with the next dose. This can be done as many times as needed to apply the full range of doses.


b. Cells in flasks or tubes are exposed to different doses of irradiation, plated in the E-Plate, and monitored using the xCELLigence System.


c. The preceding two methods can also be performed in parallel; cells are first seeded in six different E-Plates and then, at specific times, each E-Plate is exposed to different doses of radiation. The E-Plates are then monitored using the RTCA MP Instrument.


FAQ: Is it possible to use the xCELLigence System to get information about compound activity?

HelpCorner: Yes. As shown by Abbassi et al., compounds with similar activity produced similar impedance-based time-dependent cell response profiles (TCRP) [4]. The compounds were clustered based on TCRP similarity. This TCRP approach provides predictive mechanistic information for small-molecule compounds.


FAQ: Do you have any data regarding viral applications using the xCELLigence System?

HelpCorner: Yes. Please read Application Note 9 [5] and consult our publication list.


FAQ: Do you have any data on cardiomyocytes using the xCELLigence System?

HelpCorner: Yes. Please read Application Note 8 [6] and consult our publication list.


FAQ: Is it possible to coat the CIM-Plate 16?

HelpCorner: The microporous membrane of the CIM-Plate 16 can be coated on one side (electrode side) for haptotactic assays or on both sides for certain cell types in chemoattractant assays.


FAQ: Can I use primary cells with the CIM-Plate 16?

HelpCorner: Primary cells can be used with the CIM-Plate 16. We have used HUVEC cells successfully with the CIM-Plate 16; it is, however, important to use appropriate chemokines or any ECM substrates that may facilitate cell migration and attachment.

Optimization

FAQ: How can I optimize cell culture and coating conditions?

HelpCorner: To optimize your results, you may need to change one or more of the following coating and cell culture parameters:

a. Coating:

  1. Test different ECM coatings or perform a double coating.
  2. Vary the concentration.
  3. Modify the temperature used for coating
    (+15°C to +25°C, +2°C to +8°C, or +37°C).
  4. Vary the coating time (several hours to overnight).



b.  Cell culture:

  1. Change the concentration of cells: increasing or decreasing cell concentration can drastically alter results, particularly with density-sensitive cells.
  2. Be sure that the cells are healthy (e.g., number of passages, contamination, and viability).
  3. Test different concentrations of serum.
  4. Starve the cells.
  5. Modify cell detachment conditions, e.g., using EDTA detachment as opposed to enzymatic detachment.


FAQ: What should I pay attention to when planning an siRNA assay using the xCELLigence System?

HelpCorner: Several parameters must be optimized in order to achieve optimal conditions.

a. Transfection reagent:

We recommend the use of X-tremeGENE siRNA Transfection Reagent for siRNA applications. It is an optimized lipid-based transfection reagent that forms a complex with short interfering RNA (siRNA) and with mixtures of siRNA and plasmid DNA. You can find some examples of applications in Biochemica No. 2, 2006. Another powerful transfection agent, FuGENE® HD Transfection Reagent, has been successfully used for siRNA knockdown assays (Biochemica No. 1, 2008, pages 21-24) and high-throughput RNAi screening (Biochemica No. 4, 2008).



b. Mode of transfection: You can test forward or reverse transfection. In some cases, we have found that reverse transfection gives a much better phenotype compared with forward transfection.


c. siRNA concentration: You may need to perform some titration of your siRNA in order to find the optimal concentration.


d.  Seeding density: As a general rule, start with cell titration. In the ­example provided in Cell Analysis Application Note 4, 5000 cells/well were used.


FAQ: What are the optimization parameters in invasion and migration assays?

HelpCorner: The parameters to be optimized in invasion and migration assays are

a.  for invasion assay:

  1. Gel concentration
  2. Time of polymerization
  3. Temperature of polymerization
  4. Cell (number, preparation, e.g., trypsin vs EDTA)



b.  for migration assay:

  1. ECM coating type or no ECM coating
  2. Coating condition (concentration, temperature, time)
  3. Cell (number, preparation, e.g., trypsin vs EDTA)
  4. Serum starvation of the cells or not


References

  1. From Classical to Online Monitoring of G-Protein-Coupled Receptor Stimulation in Living Cells (Application Note 6).
  2. Functional Analysis of Side Effects of Transfection Reagents in the Context of Bax-induced Apoptosis (CA-Application Note 3).
  3. Using Real-Time Data from the xCELLigence System to Determine Optimal Time Points for Gene and Protein Expression Analysis (CA-Application Note 4).
  4. Kinetic cell-based morphological screening: prediction of mechanism of compound action and off-target effects. Abassi YA, Xi B, Zhang W, Ye P, Kirstein SL, Gaylord MR, Feinstein SC, Wang X, Xu X. Chem Biol 2009 Jul 31;16(7):712-23.
  5. Real-time and Label-free Monitoring of Virus-mediated Cytophathogenicity (Application Note 9).
  6. Cardiac-Specific Toxicity-Real-Time Monitoring of Averse Effects on Cardiomyocytes Derived from Embryonic Stem Cells (Application Note 8).

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