Cell-based assays that qualitatively measure the function of an interacting molecule under physiological conditions provide valuable information for drug discovery. Two approaches are commonly used in early drug discovery; target-based approaches use label-dependent molecular assays to measure the effect of compounds on a specific target protein in vitro, while phenotypic approaches use unbiased phenotypic assays to evaluate the effect of compounds on specific phenotype of cells, tissues or animals.
Since most target-based screens require either radiolabeling or fluorescent labels which may cause artifacts in results, label-free biosensors for phenotypic screens are increasingly utilized in drug discovery — These biosensors include those based on the detection of dynamic mass redistribution, and impedance biosensors using cellular dielectric spectroscopy. However, it is difficult to perform assays on suspension cells in currently available label-free methods. In addition, a relatively long incubation time is needed in order to let the cells grow to sufficient numbers for detection (e.g., ~95% confluency).
Cell-based Bio-Layer Interferometry assays (cBLI) use biosensors to capture both adherent and suspension cells. Different from current plate-based cellular assays, the bound cells on disposable Dip and Read™ biosensors have more cell surface exposed to surroundings compared to cells with the aid of gravity at the bottom of a plate well. The optical detection signal is from a combination of both reflected light and scattered light, which makes the assay capable of detecting cell response changes easily. Therefore, cBLI could potentially be beneficial to a variety of phenotypic pre-screens, not only for whole cell bindings, receptor signaling studies and cell-cell interactions, but also for lead generation and optimization, ADME and toxicity screening.
This application note discusses assay design and best practices for cell-based assays using red blood cells on the Octet system, as well as considerations for assay optimization, data acquisition and analysis. The developed cBLI methods offer users the ability to assess cell response in a versatile, high throughput, label-free, and easy-to-use format.
The Octet Plateform For Analyzing Cell Response
The Octet family of instruments utilizes optical interferometry for detection of real-time binding events without the need for labeling. Pall ForteBio’s proprietary Bio-Layer Interferometry method has been extended to measure cell binding events in real time (Figure 1). In the traditional BLI analysis of molecular interactions where the molecule sizes typically range from 150 Da all the way to 200nm, the signals collected are mainly from the interference pattern change due to reflection. However, cells are much bigger and can range from 1 to 20 micron. In this case light scattering plays an important role and the collected signals are the combined response of both the reflection interference pattern change and the light scattering change. Therefore, molecules binding to cells induces changes in cell responses (physical and physiological) which can be monitored by the Octet system. Because cells bound on biosensors have more cell surface exposed to surroundings, this can increase the assay sensitivity.
Octet instruments can read up to 96 samples simultaneously in an automated format using a standard 96-well or 384-well microplate. Cell-based assays are performed on the same biosensor platform that are utilized for biochemical assays, and are compatible with a wide range of buffers and media. The standard microplate format combined with disposable Dip and Read biosensor technology enables automated, highly parallel processing in sample volumes as low as 40 μL. Hands-on time is significantly reduced when compared with the multiple incubation and wash steps required for complex assay development with traditional cell-based assays. A wide array of biosensor specificities also enables flexibility in assay formatting.
Materials And Methods
- Octet instrument with Octet Data Acquisition and Data Analysis software
- High Precision Streptavidin (SAX) biosensors, Pall ForteBio part no. 18-5117
- For Octet RED96e instruments: 96-well, black, flat bottom, polypropylene microplate, Greiner Bio-One. part no. 655209
- Corning 3631 96-well clear-bottom plates, black, non-treated surface (Mfr #3631 – Item #UX-01728-44)
- For Octet HTX or RED384 instruments: 384-tilted well, black, flatbottom, polypropylene microplate, Pall ForteBio part no. 18-5080 or 384-well, black, flat-bottom, polypropylene microplate
- Cell Immobilization Buffer (CIB), (Pall ForteBio part no. 18-9801), for cell-capturing step ONLY
- Biosensor imaging tray, Pall ForteBio part no. 09-0206, for biosensor imaging ONLY
- Clear bottom plate: Costar 96-well black clear-bottom plate, Model# 07-200-588, for imaging analysis
- Biotinylated Wheat Germ Agglutinin (WGA), Cat No.# B-1025, VECTOR Laboratories
- Biotinylated Concanavalin A (Con A), Cat. No. B-1005, Vector Lab
- Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL I, BSL I), Cat. No. B-1105, Vector Lab
- Fibronectin (biotinylated), CatNo. FNR03-A, Cytoskeleton
- 1x PBS, Dulbecco’s phosphate buffered saline, Sigma D8662
- Advanced RPMI 1640 (1x), Gibco lifetechnologies, Ref.1263301
- Human whole blood, item code: SER-WB10ML, anticoagulant: Heparin, ZenBio (All blood products should be handled at the Bio-Safety Level 2 as recommended by the CDC/NIH manual)
Cell analysis on biosensor begins with immobilization of the cell on the biosensor surface. This example exploits the high affinity between cell capture ligands (CCL, e.g., wheat germ agglutinin) and cell membrane components (e.g., carbohydrates) for cell immobilization. The basic experiment contains six steps (Figure 2):
Step 1: Biosensor hydration to establish baseline
Step 2: Immobilization of biotinylated CCL on SAX biosensor
Step 3: Capture of the cell
Step 4: Wash and establish baseline
Step 5: Measure cell responses caused by drug treatment
Step 6: Measure cell recovery after drug treatment
Alternative methods of cell capture or immobilization, such as amine coupling, are available.
General Tips For Optimal Performance
- Fully equilibrate all reagents and samples to room temperature prior to sample preparation. Thaw frozen samples completely and mix thoroughly prior to use.
- Hydrate the biosensors for a minimum of 10 minutes prior to use. Hydrating the biosensors in a buffer consistent with the buffer used throughout the assay is recommended.
- Ensure that the Octet instrument is turned on and the lamp is warmed to room temperature for at least 60 minutes prior to starting the assay.
- Cell density recommended for optimal capture on biosensors is ~1x108/mL.
- Discard the sample if hemolysis is observed.
- Set the sample plate temperature in Octet Data Acquisition software by selecting File > Experiment > Set Plate Temperature… Enter the desired temperature. Pall ForteBio recommends running assays at 30°C, using other temperatures may require modifying the assay times discussed in this protocol. Set the default startup temperature by selecting File > Options. Enter the desired temperature under Startup.
- Cell capturing signal recommended to be >0.3 nm.
Red Blood Cell Preparation
Mix whole blood gently by inversion. Add 0.2 mL into a 1.5 mL microcentrifuge tube and centrifuge at 600 x g for 5 min. After removing an aliquot of supernatant and the top layer of the packed blood cells (but NOT the red blood cells), add 1 mL 1x PBS and mix gently by inversion. Centrifuge at 600 x g for 5 min and discard the supernatant. Repeat the wash step twice and gently resuspend the packed red blood cells with RPMI1640 to a final volume of 0.4 mL (~1x109/mL). Keep at room temperature.
Biosensor Preparation For Cell Capturing
Coating the biosensor surface with the cell capture ligand resulted in cell adherence and allowed detection of cellular responses1. An easy and quick method described below involves coupling the biotinylated cell capture ligand on SAX biosensors. All assay steps performed in the Octet system can be monitored in real time (Figure 2).
- Design the method file for capturing cells onto biosensors using basic kinetics mode in Octet Data Acquisition software:
- Set the layout with 3 steps (see example plate layout and assay steps set up in Figure 3):
- Column 1: PBS
- Column 2: biotin-CCL
- Column 3: PBS
- Design the assay steps:
- Baseline: dip the biosensors into column 1 for 60 s at 1000 rpm
- Loading of Biotin-CCL: dip the biosensors into column 2 for 120 s at 1000 rpm
- Washing: dip the biosensors into column 3 for 60 s at 1000 rpm.
- Set the layout with 3 steps (see example plate layout and assay steps set up in Figure 3):
- Pipette hydration solution (1xPBS) into the wells of a 96-well black bottom microplate (Greiner 655209) corresponding to the number and positions of the biosensors to be used.
- Hydrate the biosensors by placing the hydration plate underneath the biosensor tray containing the desired biosensors, then placing the tray on the stage inside the Octet instrument.
- Prepare biotin-extracellular matrix (20 µg/mL) and pipette into a black bottom polypropylene microplate, 96-well or 384-well depending on method design per Octet instrument. See Figure 3 for a sample plate layout using 96-well plate.
- Delay start the method for 10 min to equilibrate the sample plate on the instrument.
- Run the assay and ensure there is no significant downward baseline drift during the washing step. At least 90% of biotinextracellular matrix loading signal should remain after washing.
Cell Immobilization By Cell Capture Ligands(CCL) – CCL SCOUTING
Coating the biosensor surface with a suitable cell capture ligand is critical for a successful cBLI assay. Since the composition of CCL might vary between multicellular structures, it is recommended to screen the best CCL for the type of cell used in each individual assay. The Octet BLI platform provides an easy method for the screening. Figure 4 shows plate setup, Figure 5 is an example of the experiment layout and Figure 6 is an example of CCL scouting monitoring using human red blood cells. Four different biotinylated CCLs (20 µg/mL) were loaded on SAX biosensors and tested by using the same amount of red blood cells (~1x108/mL). Biosensor images were taken after the scouting assay (Figure 7). Results indicate the biotin-WGA performs better for red blood cell capturing, as indicated by a strong negative binding signal.
Biosensor Imaging For Cell Capture Confirmation
After the assay is completed, add 250 µL cell buffer in the clear bottom plate as shown in Figure 8 and set on the microscope stage. Put the biosensor imaging tray on top of the plate and transfer the biosensors with cell immobilized on them. Images were taken while the biosensor was kept vertical with the tip submerged in buffer using a Euromex Inverted Microscope in combination with a CMEX ImageFocus 4 digital camera.
Cell Concentration Scouting
To setup the assay, prepare several dilutions of the red blood cells in CIB (200 µL per biosensor). For example, Figure 9 shows an assay including seven cell concentrations and a negative control without loading. Prepare a sample plate according to the plate map in Figure 9 (200 µL per well). Biotinylated WGA concentration is 20 µg/mL.
In the kinetics mode of Octet Data Acquisition software, create the assay method shown in Table 1. Cell capturing can be performed at the standard running temperature of 30°C.
Hydrate the Streptavidin biosensors in immobilization buffer (1xPBS). Place the sample plate and the hydrated biosensors in the Octet instrument and start the assay, setting a delay of 600 seconds to allow the samples to equilibrate to temperature.
|Step||Data Name||Assay Time (sec)||Flow (rpm)||Step Type|
Results in Figure 10 showed that cBLI assay can detect 125 cells on the biosensor (cell number obtained by Image J). The signature of cell capturing is cell number-dependent but negative signals are expected compared to negative controls. There are two negative controls in this experiment, one is buffer only (1) and the other negative control is no B-WGA loading (2) with the highest cell loading in 8.
The observed binding data demonstrate that the cBLI signal can monitor the cell capturing on biosensor. Visualization of bound cells using the microscope helps to validate the binding event, and is not needed when the capture method has been verified. For a successful cell-based assay, >0.3 nm cell capturing signal (~500 cells) is recommended for red blood cell capturing.
Biosensor Analysis Of Cell-Drug Interactions
Ionomycin is a calcium ionophore which causes calcium influx and potassium efflux via the Ca2+ -activated potassium channel (Gardos channel), and leads to RBC shrinkage and vesiculation. Table 2 shows a suggested assay method on the Octet system for the cell response detection. Red blood cells were captured using biotinWGA and SAX biosensors as mentioned earlier, and the captured cells were exposed to a titration series of Calcium ionomycin of 10 µM, 8 µM, 6 µM and 4 µM (Figure 11).
|Step||Data Name||Assay Time (sec)||Flow (rpm)||Step Type|
|4||Binding RBC Cell||600||300||Association|
Figure 12 shows the observed results, demonstrating dose response. An initial hump was observed when the cells were dipped into Calcium ionophore ionomycin (Figure 12). All assay steps are monitored in real time.
Reproducibility Test And Example Of Data Analysis
Based on the cell responses obtained from the prior experiment, six replicates were treated with 5 µM Calcium ionomycin together with two negative controls as shown in Figure 13).
After the cell response reproducibility test completes (Figure 14), open Octet Data Analysis software and load the data folder to be analyzed. Select the biosensors to be analyzed in Step 1 of the Data Selection tab. Go to Step 2 in the Processing tab, click on the step either in the Raw Data or in the All Steps Aligned by step Association, choose the best cell response from the activation step and then in Report Points assign a time point (for example, 950 s is selected in Figure 15).
Click Add to Table and Save the file. Open the data file, a table will display (Figure 16).
|Biosensor Location||Color||Biosensor||Biosensor Type||X=950|
|Al||-1E+07||Ligand Sensor||SA (Streptavidin)||0.287|
|B1||-4E+06||Ligand Sensor||SA (Streptavidin)||0.454|
|Cl||-1E+07||Ligand Sensor||SA (Streptavidin)||0.39S|
|D1||-9E+06||Ligand Sensor||SA (Streptavidin)||0.385|
|El||-1E+06||Ligand Sensor||SA (Streptavidin)||0.436|
|Fl||-SE+06||Ligand Sensor||SA (Streptavidin)||0.447|
|Gl||-1E+07||Ligand Sensor||SA (Streptavidin)||0.038|
|Hl||-363184||Ligand Sensor||SA (Streptavidin)||0.034|
We have demonstrated in this study that red blood cells can be captured successfully on Pall ForteBio’s biosensors. The captured cells reported a measurable response when exposed to the calcium ionophore, ionomycin. Biosensor imaging provided confirmation of cell binding on the biosensor. The assay is high throughput and rapid, requiring 14 min for cell capture and 30 min for cell-drug response testing. The results indicate that cBLI can offer users the ability to assess cell response in a high throughput, label-free, versatile, and easy-to-use format.
- A novel label-free cell-based assay technology using biolayer interferometry, D. Verzijl, T.Riedl, P.W.H.I.Parren, A.F.Gerritsen, Biosensors and Bioelectronics, 87:388–395, 2017.