NCL Method ITA-8.1Chemotaxis Assay

1. Introduction

This document provides a protocol for rapid, quantitative measure of the chemoattractant capacity of a nanoparticulate material. Leukocyte recruitment is a central component of the inflammatory process, both in physiological host defense and in a range of prevalent disorders with an inflammatory component. In response to a complex network of pro-inflammatory signaling molecules (including cytokines, chemokines and prostaglandins), circulating leukocytes migrate from the bloodstream to the site of inflammation.

2. Principles

This assay represents an in vitro model, in which promyelocytic leukemia cells (HL-60) are separated from control chemoattractant or test-nanoparticles by a 3 µm filter; the cell migration through the filter is then monitored by detection of cells migrated through the filter into the bottom chamber of the culture plate using the fluorescent dye Calcein AM.

3. Reagents, Materials, Cell Lines, and Equipment

Note: The NCL does not endorse any of the suppliers listed below; these reagents were used in the development of the protocol and their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted. Please note that suppliers may undergo a name change due to a variety of factors. Brands and part numbers typically remain consistent but may also change over time.

3.1.

Reagents

3.1.1.

Phosphate buffered saline (PBS) (GE Life Sciences, Hyclone, SH30256.01)

3.1.2.

Bovine serum albumin (BSA) (Sigma-Aldrich, A4503)

3.1.3.

Fetal bovine serum (FBS) (GE Life Sciences, Hyclone, SH30070.03)

3.1.4.

RPMI-1640 (GE Life Sciences, HyClone, SH30096.01)

3.1.5.

Calcein AM (Molecular Probes, C3099)

3.1.6.

Penicillin streptomycin solution (GE Life Sciences, Hyclone, SV30010)

3.1.7.

Trypan Blue solution (Gibco, 15250-061)

3.1.8.

L-glutamine (GE Life Sciences, Hyclone, SH30034.01)

3.2.

Materials

3.2.1.

Pipettes covering the range of 0.05 to 10 mL

3.2.2.

Multi-Screen-MIC 96-well filter plates with 3µm membrane (Millipore, MAMIC3S10)

3.2.3.

Multi-Screen 96-well culture tray (feeding tray) (Millipore, MAMCS9610)

3.2.4.

96 microwell Nunclon optical bottom plates (Nunc, 165305)

3.2.5.

Polypropylene tubes, 50 and 15 mL

3.2.6.

Multichannel pipettor

3.3.

Cell Line

3.3.1.

HL-60 promyelocytic cells (ATCC, ATCC^®-CCL-240™)

3.4.

Equipment

3.4.1.

Centrifuge capable of operating at 400xg

3.4.2.

Refrigerator, 2-8ºC

3.4.3.

Freezer, −20ºC

3.4.4.

Cell culture incubator with 5% CO₂ and 95% humidity

3.4.5.

Biohazard safety cabinet approved for level II handling of biological material

3.4.6.

Inverted microscope

3.4.7.

Vortex

3.4.8.

Hemocytometer

3.4.9.

Multiwell plate reader capable of operating in fluorescence mode at 485 nm and 535 nm

4. Reagent and Control Preparation

4.1.

Complete RPMI-1640 Medium

The complete RPMI medium should contain the following reagents:

• 20% FBS (heat inactivated)
• 4 mM L-glutamine
• 100 U/mL penicillin
• 100 µg/mL streptomycin sulfate

Store at 2-8ºC protected from light for no longer than 1 month. Before use, warm in a water bath.

4.2.

Starvation Media (SM)

The starving RPMI medium should contain the following reagents:

• 0.2 % BSA
• 4 mM L-glutamine
• 100 U/mL penicillin
• 100 µg/mL streptomycin sulfate

Store at 2-8°C protected from light for no longer than 1 month. Before use, warm the medium in a water bath.

4.3.

Heat-Inactivated Fetal Bovine Serum

Thaw a bottle of FBS at room temperature or overnight at 2-8ºC and allow to equilibrate to room temperature. Incubate 30 minutes at 56ºC in a water bath, mixing every 5 minutes. Single use aliquots may be stored at 2-8ºC for up to one month or at a nominal temperature of −20ºC indefinitely.

4.4.

Positive Control

On the day of the experiment, dilute heat-inactivated FBS in serum-free medium supplemented with 0.2% BSA to a final concentration of 20%.

4.5.

Negative Control

Use PBS as a negative control. Process this control the same way as your study samples.

4.6.

Calcein AM (CAM) Working Solution

Calcein AM is supplied as a 1 mM solution. Dilute this stock solution in pre-warmed (37°C) 1X PBS to a final concentration of 4 µM (e.g. add 10 µL of stock Calcein AM to 2.49 mL of 1X PBS). The working dilution should be prepared and used ex tempore.

4.7.

Vehicle Control

Vehicle control is the buffer or media used to formulate test nanomaterials. Common excipients used in nanoformulations are trehalose, sucrose, and albumin. However, other reagents and materials are also used alone or in combination.

Vehicle control should match formulation buffer of the test-nanomaterial by both composition and concentration. This control can be skipped if nanoparticles are stored in PBS.

5. Preparation of Study Samples

This assay requires 3 mL of nanoparticles, at 1X the highest final tested concentration dissolved/resuspended in starvation medium. The concentration is selected based on the plasma concentration of the nanoparticle at the intended therapeutic dose. For the purpose of this protocol this concentration is called the “theoretical plasma concentration”. Considerations for estimating theoretical plasma concentration were reviewed elsewhere [1] and are summarized in Box 1 below.

The assay will evaluate 4 concentrations: 10X (or when feasible 100X, 30X or 5X) of the theoretical plasma concentration, theoretical plasma concentration and two 1:5 serial dilutions of the theoretical plasma concentration. When the intended therapeutic concentration is unknown, the highest final concentration is 1 mg/mL or the highest reasonably achievable concentration.

For example, if the final theoretical plasma concentration to be tested is 0.2 mg/mL, then a stock of 2 mg/mL will be prepared and diluted 10-fold (0.2 mg/mL), followed by two 1:5 serial dilutions (0.04 and 0.008 mg/mL). Use 150 μL of each of these samples per well. Each nanoparticle concentration is plated three times.

6. HL-60 Cell Preparation

HL-60 is a promyelocytic cell line derived by S.J. Collins, et al. from a patient with acute promyelocytic leukemia [2]. Cultures can be maintained by the addition of fresh medium or replacement of medium. Alternatively, cultures can be established by centrifugation with subsequent resuspension at 1x10⁵ viable cells/mL. Do not allow cell concentration to exceed 1x10⁶ cells/mL. Maintain cell density between 1x10⁵ and 1x10⁶ viable cells/mL.

6.1.

Expand cells until they are approximately 80-90% confluent (~3-5 days before experiment). Two days before the experiment, feed the cells following regular maintenance procedures.

6.2.

One day before the experiment count cells using trypan blue. If the cell viability is at least 90%, pellet cells for 5 minutes at 400xg in a 15 mL tube.

6.3.

Resuspend cells in Starvation Medium and incubate overnight (16-18 hr) at 37°C in a humidified incubator (95% air, 5% CO₂).

6.4.

On the day of experiment, count cells using trypan blue and adjust concentration to 1x10⁶ viable cells/mL in Starvation Medium.

7. Experimental Procedure

The procedure described below is based on reference 3.

7.1.

Insert the filter plate into the feeding tray and set it aside.

7.2.

Add 150 µL of starvation medium, positive control, negative control and test-nanomaterial in starvation medium into fresh feeding tray. Avoid generating bubbles while adding solutions to wells. Plate additional test-nanomaterial wells for use as cell-free controls (refer to plate map in Appendix).

7.3.

Add 50 µL of cell suspension (prepared in step 6.4) per well of the Multi-Screen filter plate (50,000 cells per well) from Step 7.1. Avoid generating bubbles while adding cells to wells. Add 50 µL of media to the cell-free control wells.

7.4.

Gently assemble Multi-Screen filter plate (step 7.3) and feeding tray containing controls and test particles (step 7.2). This is the Assay Plate. An example of the assay plate template is shown in the Appendix. Avoid shaking or tilting plates as it will disturb concentration gradient.

7.5.

Cover the plate and incubate for 4 hr at 37°C in a humidified incubator (5% CO₂, 95% air). During incubation, pre-warm PBS to 37°C and equilibrate the calcein AM to room temperature.

7.6.

Prepare a working solution of Calcein AM (CAM) as described in section 4.6 of the method.

7.7.

After the 4 hr incubation, remove the chemotaxis assay plate from the incubator. As before, avoid shaking or tilting the plate.

7.8.

Gently remove the Multi-Screen filter plate and discard it.

7.9.

Add 50 µL of 1X PBS and 50 µL of CAM working solution to appropriate wells, and 150 µL of 1X PBS plus 50 µL of CAM working solution to the reagent background control wells on the feeding tray as outlined in the example template shown in the Appendix. This is the Calcein Plate. Incubate the plate for 1 hr at 37°C.

7.10.

Transfer 180 µL of solution from the Calcein Plate to corresponding wells on a Nunc optical bottom plate and read the Nunc plate on a fluorescent plate reader at 485 nm excitation/535 nm emission.

8. Calculations

8.1.

A percent coefficient of variation (CV) should be calculated for each control or test according to the following formula:

$$\%CV=\frac{standarddeviation}{mean}\ast 100\%$$

8.2.

Background Chemotaxis

$$BackgroundChemotaxis=MeanF{U}_{SM/CAMwells}-MeanF{U}_{SM/PBSwells}-MeanF{U}_{reagentbackgroundcontrolwells}$$

8.3.

Sample Chemotaxis

$$SampleChemotaxis=MeanF{U}_{TS/CAMwells}-MeanF{U}_{SM/PBSwells}-MeanF{U}_{reagentbackgroundcontrolwells}$$

8.4.

Comparison of sample chemotaxis to background chemotaxis is performed to evaluate chemotactic potential of test material. In general, fold chemotaxis induction equal to or greater than 2 is considered positive. Statistical methods such as Student’s t-test should be applied to evaluate the significance of difference between background chemotaxis and that of test-sample.

9. Acceptance Criteria

9.1.

%CV for each control and test sample should be less than 30%.

9.2.

If two of three replicates of positive control or negative control fail to meet acceptance criterion described in 9.1, the assay should be repeated.

9.3.

Within the acceptable assay, if two of three replicates of unknown sample fail to meet acceptance criterion described in 9.1, this unknown sample should be re-analyzed.

10. References

1.

Dobrovolskaia MA, McNeil SE. Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines. J Control Release. 2013;172(2):456–66. [PMC free article: PMC5831149] [PubMed: 23742883]

2.

Gallagher R, et al. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood 54: 713–733, 1979. [PubMed: 288488]

3.

Kamath L. Multi-Screen-MIC Application note. Lit# AN1060EN00, Millipore Life Science Division.

11. Abbreviations

BSA

bovine serum albumin

CAM

Calcein AM

CV

coefficient of variation

FBS

fetal bovine serum

FU

fluorescence units

PBS

phosphate buffered saline

RBC

Reagent background control

RPMI

Roswell Park Memorial Institute

SD

standard deviation

SM

starvation media

VC

vehicle control

12. Appendix

Example Assay Plate Map

This is the template for the feeding tray. Wells in rows A-F in the upper filter plate receive 50,000 cells in 50 µL.

Row H are the cell-free test samples; they do not receive cells.

SM: Starvation Medium; NC: Negative Control; PC: Positive Control; TS: Test Sample; VC: Vehicle Control

Example Calcein Plate Map

PBS: 50 µL PBS

CAM: 50 µL Calcein AM

RBC: Reagent Background Control, 150 µL PBS + 50 µL Calcein AM

This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.

*

address correspondence to: vog.hin.liam@aniram

Potter TW, Cedrone E, Neun BW, Dobrovolskaia MA, NCL Method ITA-8.1: Chemotaxis Assay. https://ncl.cancer.gov/resources/assay-cascade-protocols DOI: 10.17917/CPWX-SP39