A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

Summary

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca²⁺ release initiated by CXCR4 activation.

Abstract

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor's function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca²⁺ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca²⁺ concentration in the absence of CXCL12) or compounds modulating the receptor's function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay's readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca²⁺.

Introduction

GPCRs are an important superfamily of cell surface proteins that activate signal transduction cascades upon extracellular ligand binding. They can be activated by a large variety of stimuli including peptides, protein hormones, biogenic amines, and lipids, which results in the initiation of diverse intracellular signaling pathways and eventually biological responses^1,2. Furthermore, GPCRs are involved in many, if not all, developmental and physiological processes and many human diseases are associated with dysfunctional GPCR signaling or receptor overexpression. GPCRs are therefore amongst the most validated pharmacological targets in medicine^1,3.

Typically, a GPCR drug discovery workflow starts with cellular screening assays enabling the identification of compounds such as small molecules, monoclonal antibodies, and peptides that can modulate the activity of a particular GPCR. In GPCR drug discovery many different types of assays exist to search for such compounds, most of which are compatible with mid- to high-throughput screening campaigns. The most used assays include receptor binding experiments, fluorescence or luminescence based assays detecting fluctuations in the level of so-called secondary messengers (e.g., Ca²⁺, cyclic adenosine monophosphate (AMP)), phenotypic screening assays, and β-arrestin recruitment assays⁴. The choice for a particular type of assay may depend on multiple factors, but is also determined by prior knowledge concerning the signaling properties of a given GPCR. Agonist binding to a GPCR induces a conformational change catalyzing the exchange of guanidine diphosphate (GDP) for guanidine triphosphate (GTP) on the α-subunit of heterotrimeric G proteins. Subsequently the G_α-GTP subunit dissociates from the G_βγ subunit and both subunits will initiate further signaling pathways. Hydrolysis of the GTP-molecule and subsequent re-association of the G_α-GDP and G_βγ subunits will restore the G protein into its inactive conformation^5,6. Based on sequence similarity of the G_α subunit different types of G proteins are defined (G_s, G_i, G_q, G_12/13)⁷. Signaling via the G_α subunit gives rise to several typical responses such as the increase (via G_s) or decrease (via G_i) of cyclic AMP production and intracellular Ca²⁺ mobilization (via G_q)^5,7. G_βγ subunits are also able to induce intracellular effector pathways. For instance, upon activation of G_i-coupled GPCRs, G_βγ can directly stimulate phospholipase C (PLC-β) to produce inositol triphosphate (IP₃) that triggers the release of Ca²⁺ from intracellular stores⁷. Following receptor activation, GPCRs are phosphorylated by GPCR kinases (GRKs) which promotes interaction with β-arrestins. This process terminates G protein signaling and leads to receptor desensitization and eventually internalization. β-arrestins are also able to form multi-molecular complexes that can trigger other signaling pathways independent of G protein signaling⁸.

Within the subfamily of chemokine receptors, the G_i-coupled CXCR4 is a GPCR that has raised much interest as a promising target for drug discovery. Given its established role as a major co-receptor for human immunodeficiency virus 1 (HIV-1) viral entry and infection, compounds targeting CXCR4 were initially developed as anti-HIV drug candidates⁹. More recently, a growing body of evidence has pointed to an important role for CXCR4 in tumorigenesis and cancer metastasis making it a well-validated therapeutic target in oncology as well¹⁰. CXCR4 is highly expressed in more than twenty types of human cancer and controls tumor cell survival, proliferation, and migration as well as tumor-related angiogenesis¹⁰. CXCR4 antagonists of different chemical classes have previously been described^11,12, but only the small molecule AMD3100 is currently approved for use in the clinic as a stem cell mobilization agent used during treatment of lymphoma and myeloma patients^13,14. Clinical trials are ongoing to evaluate the safety and efficacy of several other CXCR4 antagonists in different human diseases, but with a strong focus on oncology¹². Given the many potential applications for CXCR4 antagonists, the search for novel compounds with improved pharmacokinetic properties, improved bioavailability, or potentially less side effects is warranted.

Herein, a kinetic fluorescence-based cellular assay primarily used to screen for compounds capable of inhibiting CXCR4 is described. The fluorescent measurement of this method is based on the transient increase of the intracellular Ca²⁺ concentration evoked upon CXCR4 activation by its endogenous agonist, the chemokine ligand CXCL12 (formerly known as stromal cell derived factor 1α (SDF1-α)), and the potential inhibition of this CXCL12-induced Ca²⁺ response by particular compounds. In this assay, U87 human glioblastoma cells stably expressing the human CXCR4 receptor are used. At the same time, these cells lack endogenous expression of CXCR7, a related chemokine receptor that also binds CXCL12^15,16,17. CXCR7 has previously also been shown to be capable of forming heterodimers with CXCR4, thereby modulating the signaling properties of this latter receptor¹⁸. Fluctuations in the level of intracellular Ca²⁺ mediated by CXCR4 are monitored by loading the CXCR4⁺ cells with fluo-2 acetoxymethyl (AM) ester, a cell-permeable high affinity fluorescent Ca²⁺-binding dye. Fluo-2 AM is a single wavelength fluorescent molecule that can be excited at 490 nm while its emission fluorescence is measured at 520 nm. This emission fluorescence increases upon Ca²⁺ binding, with a large dynamic range between the Ca²⁺-bound and unbound state. The increase in fluorescent signal is transient, occurring within a time interval of a few minutes, and will decay afterwards. The peak height of the fluorescent emission further correlates with the level of receptor activation. The assay itself is performed using a fluorescence microplate reader equipped with an Intensified CCD (ICCD) camera that possesses an integrated pipetting system that allows standardization of the pipetting steps in the assay (see Table of Materials). In addition, the simultaneous measurement of the fluorescent signal in all wells of a microplate is another key advantage of the fluorescence reader that is used. During the first part of the assay the compounds under investigation (e.g., a panel of small molecules at a fixed concentration or in a dilution series) are added to the fluo-2 AM loaded CXCR4⁺ U87 cells followed by a ~ 10 min incubation period during which the potential agonistic effect of the compounds is continuously measured in real time. Then, the endogenous agonist (i.e., CXCL12) is added to the cells to evoke a CXCR4-mediated transient increase in the level of intracellular Ca²⁺. During this part of the assay the potential antagonistic activity of the tested compounds can be evaluated. A schematic overview of the assay's general workflow is presented in Figure 1.

Although this Ca²⁺ mobilization assay has primarily been used to identify and determine the inhibitory potency of competitive CXCR4 antagonists (i.e., compounds that prevent the endogenous agonist to bind and stimulate the receptor), it also can identify receptor agonists and, in addition, compounds that exert their function by binding at allosteric sites (i.e., sites that topographically differ from the orthosteric binding site occupied by the endogenous agonist). Examples of the latter category of compounds include allosteric agonists and PAMs and NAMs^19,20. Whereas receptor-specific antagonists as well as NAMs would inhibit the CXCL12-induced Ca²⁺ response, PAMs would enhance this response (see also Discussion section). Although the assay described herein specifically targets CXCR4, it is anticipated that this method can be applied to other GPCRs with minimal optimization effort, at least if they signal via the release of intracellular Ca²⁺.

Protocol

NOTE: All steps described under sections 1 and 2 are carried out under sterile conditions in a laminar flow cabinet.

1. Maintenance of U87.CD4.hCXCR4 Cells

1. Grow the cells in T75 culture flasks at 37 °C and 5% CO₂ in a humidified incubator.
   NOTE: The in vitro cell line used in this protocol is a U87 glioblastoma cell line stably expressing cluster of differentiation 4 (CD4) and human CXCR4 and has been previously described¹⁷. Cell surface expression of CD4 and CXCR4 is continuously monitored by flow cytometry and expression levels remain constant over time (~ 100% CD4⁺ and ~ 100% CXCR4⁺ cells). A detailed description of the generation of the used cell line and of the flow cytometry procedure to investigate receptor expression levels is not within the scope of this protocol.
   1. Subculture cells at 80-85% confluency. Allow all reagents to reach room temperature (RT) before cell culturing.
   2. Remove the conditioned culture medium from the cells and wash the cell monolayer once with 5 mL phosphate buffered saline (PBS).
   3. Add 3 mL of 0.25% trypsin-EDTA and distribute it evenly over the cell monolayer. Then remove the excess of trypsin-EDTA and incubate up to 5 min at 37 °C until cells start to detach.
   4. Add 10 mL of fresh complete growth medium (Dulbecco's Modified Eagle's Medium (DMEM) + 10% Fetal Bovine Serum (FBS) + 0.01 M HEPES + appropriate selection agents). Resuspend the cells by gently pipetting up and down. Transfer the cell suspension to a sterile 50 mL tube.
   5. Count the number of viable cells using a method of choice. In the case of U87 glioblastoma cells, viability generally reaches ~ 100%.
      NOTE: Cell numbers can be determined in several ways. We routinely use an automated cell analysis system based on trypan blue staining (see Table of Materials) according to its standard handling procedures, but other manners should work equally well.
   6. Add 2 x 10⁶ or 3 x 10⁶ (viable) cells to a final volume of 25 mL fresh growth medium in a T75 culture flask and incubate at 37 °C and 5% CO₂.
      NOTE: If 3 x 10⁶ cells are used the cells will reach 80-90% confluency after 2 days. If 2 x 10⁶ cells are used, they will reach the same confluency after 3 days. The growth rate of the cells should first be determined empirically if other cell lines are used.

2. Seeding of the Cells for the Ca²⁺ Mobilization Assay

1. At the day of cell passaging (day 0), coat black-walled polystyrene 96-well plates with clear bottom (see Table of Materials) with a 0.1% gelatin solution to facilitate cell attachment.
   NOTE: Coating of the 96-well plates with gelatin might be omitted if pre-coated plates, which are commercially available (see Table of Materials), are used. It is recommended to evaluate the use of pre-coated plates instead of manual coating for each cell line under investigation before continuing with the protocol.
   1. Prepare gelatin solution by adding 1 g of gelatin to 100 mL PBS to obtain a 1% solution. Dilute by 10x with PBS before further use. To improve the solubility of gelatin, heat the solution to 37 °C.
   2. Add 100 µL of 0.1% gelatin solution per well of the 96-well plate using a multichannel pipette. Incubate for 2 h at RT.
2. In the meantime, prepare the cells for seeding in the coated 96-well plate.
   1. Detach the cells from the culture flask, resuspend them in fresh growth medium, and count them using trypan blue staining method.
   2. Spin down the cells in a 50 mL tube for 5 min at 400 x g at RT.
   3. Resuspend the cells in fresh growth medium (DMEM + 10% FBS + 1% HEPES, no antibiotics) to obtain a cell density of 0.1 x 10⁶ (viable) cells/mL.
3. Remove the gelatin solution from the black-walled plates with clear bottom by flipping over the plate and drying it on a tissue. Add 200 µL/well PBS to remove excess gelatin and flip over the plate again.
4. Dispense 200 µL of the cell suspension (corresponding to 0.2 x 10⁵ cells) per well in the gelatin-coated 96-well plate (from now one this plate will be further referred to as the "measurement plate").
5. Incubate the plate overnight at 37 °C and 5% CO₂.

3. Loading of the Cells with a Fluorescent Ca²⁺ -sensitive Dye

1. At day 1 perform the actual Ca²⁺ mobilization assay, starting with loading of the seeded cells with the fluorescent Ca²⁺-binding dye fluo-2 AM.
   1. Prepare assay buffer by adding 40 mL HEPES (1 M) to 200 mL Hank's Balanced Salt Solution (HBSS, 10x, no phenol red, no sodium bicarbonate). Add ultrapure water to obtain a final volume of 2 L and add 4 g Bovine Serum Albumin (BSA). Dissolve the BSA via magnetic stirring. Adjust the pH to 7.4 (with NaOH) and filter the solution.
      NOTE: During all following steps the same buffer, referred to as "assay buffer", is used.
   2. Prepare a stock solution (4 mM in dimethyl sulfoxide (DMSO)) of the fluorescent Ca²⁺-sensitive dye fluo-2 AM. Avoid excessive exposure to light. Dissolve 1 mg fluo-2 AM (molecular weight: 1,061 g/mol) in 235.6 µL DMSO.
   3. Prepare a working solution of fluo-2 AM. For one 96-well assay plate mix 12.5 µL of fluo-2 AM stock solution (4 mM) and 12.5 µL nonionic surfactant polyol (e.g., pluronic f-127) solution (20% weight/volume in DMSO, see Table of Materials). Add 22 µL of this mixture to 11 mL assay buffer in a 15 mL tube. The final concentration of fluo-2 AM is 4 µM.
      NOTE: The nonionic surfactant is used as a dispersion agent to improve the aqueous solubility of fluo-2 AM and, in consequence, to enhance cell loading. To solubilize surfactant in DMSO, heat the sample at 37 °C and mix regularly to obtain a homogenous solution.
   4. Remove the growth medium from the previously seeded cells in the 96-well measurement plate by flipping over the plate and drying it on a paper towel.
   5. Add 100 µL of loading dye solution per well using a multichannel pipette and incubate for 45 min at RT in the dark.

4. Preparation of 96-well Polypropylene Plates Containing the Chemokine Ligand CXCL12 or the Compounds under Investigation

1. Obtain round bottom polypropylene (PP) 96-well plates (see Table of Materials).
   1. Allow the stock solution of CXCL12 (1 mg/mL) and the stock solution of the compounds under investigation to equilibrate at RT.
      NOTE: The stock solution of CXCL12 is prepared in ultrapure water supplemented with 0.01% Tween20 and stored at -20 °C as single-use aliquots.
   2. Prepare a 5x concentrated solution of CXCL12 in assay buffer (250 ng/mL which equals 31.25 nM) and a 5x concentrated dilution of each compound (in assay buffer).
   3. Dispense the stock solutions into the appropriate 96-well plate according to a predefined plate layout. Dispense 75 µL/well in the plate containing CXCL12 (the "chemokine plate"), dispense 50 µL/well in the plate containing the compounds (the "compound plate").
      NOTE: Both compounds and CXCL12 will finally become diluted 5x upon dispensing in the measurement plate. It is also important to include negative control wells containing only assay buffer in both the compound plate as well as the chemokine plate. Also positive control wells need to be included (i.e., wells with CXCL12 in the chemokine plate, but with assay buffer in the corresponding wells of the compound plate).

5. Protocol Settings on the Fluorescence Microplate Reader

NOTE: The fluorescence microplate reader used in this protocol is referred to in the Table of Materials.

1. Switch on the cooler unit of the fluorescence plate reader first, and then switch on the fluorescence reader itself. Let initiate for a few minutes and open the system's software.
2. Create the assay's protocol using the drag-and-drop menu that includes the following steps:
   1. In the "Settings" box, select 'Read_Mode' with excitation wavelength: 470-495 nM; emission wavelength: 515-575 nM.
      NOTE: The selected wavelengths are appropriate for use with the Ca²⁺-sensitive dye fluo2.
   2. Include the "Mix with TF" (transfer fluid) box for automatic mixing of the compounds in the compound plate, that will be put at the source 2 position of the device (see step 6.4). Select 15 µL of solution in each well to be automatically aspirated and mixed three times. Adjust the height of the pipette tips at 20 µL below the liquid surface. Set the speed of aspiration and dispensing at 50 µL/s.
      NOTE: The height of the pipette tips refers to the volume that is left underneath the pipette tips. For instance, if the wells of a compound plate contain 50 µL, a height of 30 µL corresponds to 20 µL below the liquid surface. The volume of compound solution taken to mix, the speed of mixing, the position of the pipette tips during mixing, and the number of mixing cycles can all be adjusted using the software.
   3. In the "Transfer Fluid" box, define that 20 µL of each well from the compound plate will be transferred into the measurement plate. Position the pipette tips at 20 µL below the liquid surface i.e., at height 30 µL during aspiration of the compounds and at height 60 µL during dispensing in the measurement plate. Set the speed of aspiration at 50 µL/s and the speed of dispensing at 25 µL/s.
      NOTE: The compound plate contains 50 µL of solution per well (see step 4.1.3), thus a height of 30 µL corresponds to a position 20 µL below the liquid surface. The measurement plate will contain 80 µL of assay buffer per well (see step 6.3), thus a height of 60 µL corresponds to a similar position of the tips.
   4. Select the "Read with TF" button. During the first interval, select 60 reads (fluorescent measurements) with a read interval time of 1 s. Define that 10 reads are recorded before dispensing of the compounds into the measurement plate, and 50 reads afterwards. Define the second interval with a read interval time of 30 s and 18 reads.
      NOTE: During this step fluorescence will be measured kinetically (at the defined time intervals) for ~ 10 min in total.
   5. Include the "Wash Tips" button in the protocol three times. Within each washing step, select the fluid type: fluid A (ultrapure water) or fluid B (70% ethanol), the number of wash cycles (1), the pump speed (Fast), and the number of strokes (5) during each wash step.
      NOTE: During the first and last wash step fluid A is used, during the second wash step fluid B is used.
   6. Include the "Pause Pipettor" function. Set the pipettor to pause for 300 s.
      NOTE: By including this step in the protocol, the pipettor head, which is integrated in the fluorescence plate reader device, will pause for 5 min before continuing the protocol.
   7. Include the "Mix with TF" box to allow automatic mixing of the CXCL12 solution in the chemokine plate, that will be positioned at the source 3 position of the device (see step 6.4). Select 15 µL of solution in each well to be automatically aspirated and mixed three times. During aspiration and mixing, position the pipette tips 20 µL below the liquid surface. The speed of aspiration and dispensing is set at 50 µL/s.
      NOTE: Similar as for step 5.2.2, these parameters can be adjusted.
   8. In the subsequent "Transfer Fluid" box, define that 25 µL from each well of the chemokine plate will be transferred into the measurement plate. Position tips 20 µL below the liquid surface i.e., at height 55 µL during aspiration from the chemokine plate that contains 75 µL/well (see step 4.1.3), and at height 80 µL during dispensing in the measurement plate.Set the speed of aspiration at 50 µL/s and the speed of dispensing at 25 µL/s.
   9. Include the "Read with TF" button. During the first interval, select 145 reads with a read interval time of 1 s. Define that 5 reads are recorded before dispensing CXCL12 into the measurement plate, and 140 reads afterwards. Define the second interval with a read interval time of 6 s and select 20 reads.
      NOTE: Taken together, throughout the entire protocol 243 reads (fluorescent measurements) are recorded: 78 at step 5.2.4 and 165 during step 5.2.9.
   10. Similar to step 5.2.5, include the "Wash tips" button three times in the protocol.
       NOTE: Including this step at the end of the protocol allows that the tips can be re-used to perform another assay without needing to change the tips.
3. Set the temperature of the device at 37 °C by clicking the "Set stage temperature" button and selecting 37 °C.

6. Running the Fluorescence Assay

1. After the measurement plate has incubated with loading buffer for 45 min (see step 3.1.5), remove the buffer by flipping over the plate and dry it on a tissue.
2. Wash the seeded cells by adding 150 µL/well of assay buffer and incubate for 2 min.
3. Remove the buffer again by flipping over the plate. Add 80 µL/well of assay buffer with a multichannel pipette.
4. Put all the plates into the device at their appropriate position: the compound plate at the source 2 position, the chemokine plate at the source 3 position, and the measurement plate at the read position. Put a box of black tips at the source 1 tips position. Shut the device's door and incubate for 5 min before continuing the protocol.
5. Select the "Protocol signal test" button to determine the background relative light units (RLUs) and fluorescence variance over the plate. A new window will pop up. Select "Test signal".
   NOTE: During this step, background RLUs are determined by the ICCD camera, which is integrated in the optics compartment of the fluorescence reader device. These RLUs result from the excitation of the Ca²⁺-sensitive dye (fluo-2) by light emitting diodes (LEDs) (LED output wavelength 470-495 nm). Values of 8,000-10,000 RLUs are well suited for this application. RLUs can, if necessary, be adapted by changing the excitation intensity (Select Exc. Intensity), or the camera gate (Select Gate Open). The variance over the plate should ideally be less than 7.5%.
6. Select "Update" if background values of 8,000-10,000 RLUs are obtained. Save the main protocol by clicking the Save button.
   NOTE: By doing so, the settings from the protocol signal test will be carried over to the main protocol.
7. Run the assay by pushing the "RUN" button.

7. Data Analysis and Quality

1. After the assay has finished, open the "Analysis" box in the system's software.
   1. Go to "configure corrections" and choose "response over base line". Define base line 1 to start at measurement 1 and end at measurement 5; the mean fluorescence will be calculated in each well between measurement 1 and 5.
      NOTE: All further measurements from a particular well are divided by this well-specific base line 1.
      1. Define base line 2 to start at measurement 78 until measurement 83 (CXCL12 is dispensed after measurement 83; again the mean fluorescence is calculated in each well between measurement 78 and 83 and this correction factor is applied to all measurements following measurement 83).
   2. Activate the tick box "show as percentage" and "subtract background".
   3. Go to "Configure Kinetic Reduction". To evaluate the inhibitory effect of compounds on the CXCL12-induced Ca²⁺ response, choose Max-Min starting from measurement 84 (i.e., the first measurement after which CXCL12 is dispensed) to 243 (i.e., the final measurement).
      NOTE: By choosing Max-Min the difference between the minimum and maximum response over baseline between measurement 84 and measurement 243 is reported. If the Max-Min is chosen starting between measurement 11 and measurement 78, the difference between the minimum and maximum response over baseline relative to baseline 1 is reported. This value can be used to analyze the potential agonistic activity of the compounds, see Discussion).
   4. The data will be visualized in the system's software. If needed, export the raw data for additional analysis and visualization in other common analysis software packages.

Results

The effect of CXCL12 stimulation on the intracellular Ca²⁺ mobilization in U87.CD4.CXCR4⁺ and U87.CD4 cells was evaluated with the Ca²⁺ mobilization assay. Instead of 20 µL of test compound that would normally be added during the first pipetting step of the protocol (Figure 1), assay buffer was added to the fluo-2 AM loaded U87.CD4.CXCR4⁺ cells in the measurement plate. During the second dispensing step, different c...

Discussion

The Ca²⁺-mobilization assay described herein has previously been shown to be a valuable tool to identify and characterize receptor antagonists targeting CXCR4¹⁷. It is, however, anticipated that this method can be more generally applied to a large group of other GPCRs that trigger a cytosolic Ca²⁺ release upon their activation, as illustrated for the related chemokine receptor CCR5. Whereas in the case of CCR5 exactly the same experimental conditions could be applied, several...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluo-2 AM	Abcam	ab142775	fluorescent Ca2+ sensitive dye
Pluronic F-127	Sigma	P2443-250G	pluronic acid
Gelatin	Sigma	G9391
AMD3100	Sigma	A5602-5 mg	specific CXCR4 antagonist
Maraviroc	kind gift of AnorMed		antiretroviral drug, CCR5 antagonist
Chemokine ligand CXCL12	PeproTech	300-28A
Chemokine ligand CCL5	PeproTech	300-06
Fetal Bovine Serum (FBS)	Gibco (Life Technologies)	10270-106
Bovine Serum Albumin (BSA)	Sigma	A1933-25G
Dulbecco's Modified Eagle's Medium (DMEM)	Gibco (Life Technologies)	41965-039
HBSS (10 x), calcium, magnesium, no phenol red	Gibco (Life Technologies)	14065-049
HEPES (1 M)	Gibco (Life Technologies)	15630-056
Trypsin-EDTA (0,25 %), phenol red	Gibco (Life Technologies)	25200-056
Dulbecco's Phosphate Buffered Saline (DPBS)	Gibco (Life Technologies)	14190-094
Falcon tubes, 50 mL	Greiner Bio-One	227 261
Tissue culture flask (T75)	Corning	353024
Black plate, 96-well, clear bottom, with lid	Costar/Fisher Scientific	10530753	assay plate (96-well), for cell seeding
Polypropylene (PP) plates	Thermo Scientific (VWR)	732-2661	plates used to prepare the compound plates and chemokine plates, round bottom
FLIPR Tetra high throughput cellular screening system	Molecular Devices		Fluorescent plate reader with integrated pipettor head and ICCD camera
FLIPR Tetra LED Module 470 - 495 nm	Molecular Devices	0200-6128	Light emitting diodes for excitation of the fluorescent Ca2+ sensitive dye
FLIPR Tetra Emission Filter 515 - 575 nm	Molecular Devices	0200-6203	emission filter compatible with the fluorescent dye
FLIPR Tetra 96 Head	Molecular Devices	0310-4536	96-well pipettor head, integrated within the fluorescent plate reader
ScreenWorks	Molecular Devices		software package used for data analysis and visualization on the FLIPR Tetra
Vi-CELL	Beckman Coulter		cell viability analyzer
Corning CellBIND 96 Well Flat Clear Bottom Black Polystyrene Microplates, with Lid, Sterile	Corning	3340	Pre-coated 96-well assay plates that may represent an alternative for manual coating of the assay plate.