Chemotaxis Bioassay

Introduction

R&D Systems uses chemotaxis bioassays to measure the activity of the following chemokines and neutralizing antibodies:

Mouse Chemokines

6Ckine	Eotaxin	MARC	MIP-3 beta
C10	Fractalkine	MCP-5	MPIF-2
CCL28	I-TAC	MIG	RANTES
CRG-2	JE	MIP-1 alpha	SDF-1 alpha
CTACK	Lymphotactin	MIP-1 beta	TCA-3

Chemotaxis Bioassay Materials Cell Preparation Alcohol 2X PBS 1X PBS Percoll (Amersham Pharmacia BioTech Catalog # 17-0891-01) RPMI 1640 (Irvine Catalog # 9160), supplemented 2 mM L-glutamine 100 U/mL Penicillin 100 mg/mL Streptomycin DMEM high glucose (Irvine Catalog # 9024),supplemented 2 mM L-glutamine 100 U/mL Penicillin 100 mg/mL Streptomycin Fetal Bovine Serum (Sigma Catalog # F2442) G-418 (Gentamycin sulfate) (Sigma Catalog # G1264) 2-mercaptoethanol Ficoll-Hypaque M (1.119 mg/mL final density) Ficoll type 400 (Sigma Catalog # F4375) Hypaque M-76 (Nycomed Catalog # NDC 0407-0776-04) Make a 9% Ficoll type 400 solution with ddH20 Combine 900 mL 9% Ficoll + 200 mL Hypaque M-76 Sterilize Check density Cell Growth Fresh lymphocytes: purified from fresh blood or leukocyte units - use to test rhFractalkine, rhMPIF-1, rhPARC, rmFractalkine Growth Medium: RPMI 1640, supplemented 10% Fetal Bovine Serum (Sigma Catalog # F2442) Cultured lymphocytes: purified from fresh blood or leukocyte units and cultured - use to test rh6Ckine, rhCXCR4, rhMIP-3 beta, rhSDF-1 alpha, rhSDF-1 beta, rm6Ckine, rmMIP-3 beta, rmSDF-1 alpha Growth Medium: RPMI 1640, supplemented 10% Fetal Bovine Serum (Sigma Catalog # F2442) 50 µM 2-mercaptoethanol IL-2 Cultured lymphocytes: purified from fresh blood or leukocyte units and cultured - use to test rhCXCR3, rhI-TAC, rhIP-10, rhMDC, rhMIG, rmCRG-2, rmI-TAC, rmLymphotactin, rmMIG Growth Medium: RPMI 1640, supplemented 10% Fetal Bovine Serum (Sigma Catalog # F2442) 50 µM 2-mercaptoethanol 10 ng/mL rhIL-2 (R&D Systems Catalog # 202-IL) Monocytes: purified from fresh blood or leukocyte units and cultured 2 days to allow detachment from flask - use to test rhHCC-1, rhMCP-1, rhMCP-2, rhMCP-3, rhMCP-4, rhMIP-1 alpha, rhMIP-1 beta, rhRANTES, rmC10, rmJE, rmMARC, rmMCP-5, rmMIP-1 alpha, rmMIP-1 beta, rmRANTES Growth Medium: RPMI 1640, supplemented 10% Fetal Bovine Serum (Sigma Catalog # F2442) Y3-hCCR3 Cells: human CCR3 transfected rat myeloid cell line obtained from Dr. Albert Zlotnik at DNAX - use to test rhEotaxin, rhEotaxin-3, rhMPIF-2, rmEotaxin, rmMPIF-2 Growth Medium: DMEM, supplemented 10% Fetal Bovine Serum (Sigma Catalog # F2442) 25 mM HEPES (GIBCO Catalog # 15630-080) 1 mg/mL G-418 (Sigma Catalog # G1264) BW5147.G.1.4.OUAR.1 Cells: AKR/J mouse T cell lymphoma cell line, ATCC Catalog # CRL-1588 - use to test rhI-309, rmTCA-3. TIB-48 cells (BW5147.G.1.4 thioguanine resistant ATCC cell line) can also be used Growth Medium: DMEM, supplemented 10% Fetal Bovine Serum (Sigma Catalog # F2442) mCCR10 Transfected BaF/3 Cells: mouse CCR10 transfected, IL-3 dependent cell line derived from mouse B cells - use to test rhCCL28, rhCTACK, rmCCL28, rmCTACK Growth Medium: RPMI 1640, supplemented 10% Fetal Bovine Serum (Sigma Catalog # F2442) 50 µM 2-mercaptoethanol 10 ng/mL rmIL-3 (R&D Systems Catalog # 403-ML) 10 mM HEPES (GIBCO Catalog # 15630-080) 1 mg/mL G-418 (Sigma Catalog # G1264)

Procedure

Cell Growth and Preparation
As with all materials of human source, gloves and lab coats should be worn. All materials contaminated by these cells should be either decontaminated or disposed of in biohazard containers to be autoclaved. All procedures are carried out under sterile conditions.

1. Obtain fresh human heparinized blood.
2. Using 1 part of Ficoll-Hypaque M, carefully pipette 2 parts of heparinized blood on top of the Ficoll-Hypaque M in a 15 mL centrifuge tube.
   1. Centrifuge 20 minutes at 500 x g, 20 °C.
      Do not use centrifuge brake.
   2. Carefully remove tubes from centrifuge. There should now be very distinct layering of the blood. The very top layer should be pale yellow and is the plasma portion of blood. The mononuclear cells (MNCs contain lymphocytes and monocytes) are contained in the band just under the plasma which also contains platelets. This band separates the plasma from the ficoll layer. The polymorphonuclear cells (PMNs contain granulocytes, eosinophils and basophils) are suspended in the ficoll-hypaque layer, RBCs make up the lowest layer.
3. Carefully draw off MNC layer with a plastic pipette, taking some plasma but no PMNs.
4. Draw off PMNs into another tube. Avoid taking any RBCs (these can be used in the myeloperoxidase assay, for neutrophil chemotaxis or eosinophil chemotaxis).
5. Wash MNC's with PBS.
1. Dilute cells at least 1:4 with 1X PBS and centrifuge 5 minutes at 500 x g.
2. Repeat this wash until the supernatant is clear and all platelets are removed.
3. Count cell number and obtain a differential identifying the types and percentage of cells in your prep. There should be <10% granulocytes and no platelets.
6. Resuspend cells in appropriate Growth Medium to 10 x 10⁶ cells/mL and add to a tissue culture flask. Incubate for one hour at 37 °C. Remove suspended cells (lymphocytes, which are processed further in step 7) and add fresh media to more than cover attached cells in the flask (monocytes). Allow cells to incubate for 48 hours at 37 °C. The monocytes should now be in suspension and are ready to use in the monocyte chemotaxis assay.
7. Centrifuge lymphocytes. These are now ready to be used in the Fresh Lymphocyte Chemotaxis. For Cultured Lymphocyte Chemotaxis, resuspend to 2 - 3 x 10⁶ cells/mL in appropriate Growth Medium. Incubate for 5 - 8 days (for cultured lymphocytes) or 3 weeks (for IL-2 cultured lymphocytes) prior to using in chemotaxis. Replace media when cells are overcrowded and media spent. The media for the IL-2 cultured lymphocytes will need to be changed every week to replenish nutrients and remove waste.
8. Larger numbers of lymphocytes/monocytes can be obtained through the purchase of leukocyte units from your community blood center. Cell population separation must then be performed using a pre-formed Percoll gradient and will not occur using the above Ficoll-Hypaque M. Refer to the section below on cell purification from leukocyte units.
9. Lesser amounts of cells need to be purified if the 48-well microchemotaxis chamber is used. Detection of cell chemotaxis is accomplished by counting the number of cells migrated per well through a microscope (usually this is the average count per 4 - 5 high power fields).
   Purification of Monocytes and Lymphocytes from

1. Cut the alcohol tube protruding from blood unit with a pair of scissors and allow blood to flow into a sterile 50 mL centrifuge tube.
   Units must be at room temperature.
   Whenever more than one unit of blood is received, process each unit separately.
2. Centrifuge 20 minutes at 500 x g.
3. Three distinct layers are formed during centrifugation. The top layer is plasma. The middle layer is white blood cells and the bottom layer consists of red blood cells. Remove and discard most of plasma layer. Transfer WBC layer and top of RBC layer to a 50 mL centrifuge tube and dilute to 50 mL with 1X PBS. Remaining RBC layer can be discarded.
4. To concentrate the WBC layer, repeat centrifugation procedure 3 or 4 times. This will remove the majority of RBCs. Remove platelets from the preparation by centrifuging for 10 minutes at 1600 rpm. Remove and discard supernatant only. Repeat this procedure 3 or more times until platelets no longer cause the supernatant to be cloudy.
5. Meanwhile, prepare Percoll/PBS gradient tubes: Sterilize and dry 15 mL polycarbonate Sorvall centrifuge tubes using alcohol. Add to each tube 3.5 mL of 2X PBS and 4 mL of Percoll (Add sequentially in any order. The two solutions may mix during addition, but do not purposely mix). Centrifuge for 40 minutes at 16000 rpm using a Sorvall SS-34 rotor.
6. Resuspend WBC pellet with 1X PBS (resuspend in only enough volume to add to all tubes made) and carefully overlay 2 mL of suspension onto pre-formed Percoll gradients.
7. Centrifuge for 20 minutes at 2000 rpm.
8. Four distinct layers are formed during centrifugation. From top to bottom they are platelets, monocytes, lymphocytes/monocytes, granulocytes/red cells. Carefully remove and discard the platelet layer. Remove and transfer monocyte layer of each Sorvall tube to a labeled 50 mL tube. Remove and transfer the lymphocyte/monocyte layer of each Sorvall tube to another labeled 50 mL tube. Discard granulocytes/red cells.
9. Dilute both cell preparations with 1X PBS to 50 mL. Remove a 0.5 mL aliquot and count cells to determine cell number and differential.
10. Remove any remaining platelets from cell preparations by centrifuging suspension for 5 - 10 minutes at 1600 rpm. Repeat procedure 2 or more times.
11. Based on cell count, resuspend cell pellet to 2 - 4 x 10⁷ cells/mL in appropriate Growth Medium and seed T-175 flasks (1 each for monocyte and lymphocyte/monocyte preparations).
12. Allow cells (monocytes) to attach to flask. Incubate for 1 hour at 37 °C in a 5% CO2 humidified chamber. Gently pour off suspended lymphocytes into another flask (continue with step 13) and add back appropriate Growth Medium. Return flask to incubator and use cells in monocyte chemotaxis assay 2 days later when monocytes begin growing in suspension.
13. Centrifuge lymphocytes. These are now ready to be used in the Fresh Lymphocyte Chemotaxis. For Cultured Lymphocyte Chemotaxis, resuspend to 2 - 3 x 10⁶ cells/mL in appropriate Growth Medium. Incubate for 5 - 8 days (for cultured lymphocytes) or 3 weeks (for IL-2 cultured lymphocytes) prior to using in chemotaxis. Replace media when cells are over crowded and media spent. The media for the IL-2 cultured lymphocytes will need to be changed every week to replenish nutrients and remove wastes.
    

1. Seed cells at 5 x 10⁴ cells/mL in appropriate Growth Medium.
2. Split cells every 3 - 4 days and re-seed in fresh media.
3. Use cells when they have just become confluent and have had fresh media added within the past 24 - 48 hours.

1. Seed cells at 5 x 10⁴ cells/mL in appropriate media.
2. Split cells every 3 days and re-seed in fresh media.
3. Use cells when they have just become confluent and have had fresh media added within the past 24 - 48 hours.
   

1. Seed cells at 5 x 10⁴ cells/mL in appropriate Growth Medium.
2. Split cells every 3 - 4 days and re-seed in fresh Growth Medium.
3. Use cells when they are in log phase and have had fresh media added within the past 24 hours.
   

1. Make serial dilutions of samples in Assay Medium.
1. Add 100 µL Assay Medium to each well of 96-well plate.
2. Leave column 12 as a blank (cells + media only control).
3. Add 50 µL of sample or standard to appropriate row of column 1. Serially dilute by mixing the 50 µL 4 - 5 times and transferring 50 µL to next well in row. Repeat to column 11. Remove and discard last 50 µL.
4. Set up antibodies as per package insert; preincubate serial dilutions of the antibody with cytokine (at a concentration where the final concentration is at maximal chemotaxis). Cytokine only and antibody only control wells are required.
5. Antibodies to cell surface receptors are set up with constant amount of cytokine in lower chamber at a final concentration to be at maximal chemotaxis and addition of serially diluted antibody to the top chamber with cells
2. Transfer 75 µL of volume from sample plate to appropriate wells of the 96-well chemotaxis chamber. See manufacturer's instructions on assembly, care and cleaning of these chambers. A clean chamber is important in obtaining good results.
3. Assemble chamber as per manufacturer's instructions using the 5 µm pore size PVP-free polycarbonate filter (8 µm for monocyte chemotaxis). Avoid forming bubbles as they will cause variation in reading the plate.
4. Resuspend cells to 20 x 10⁶ cells/mL (10 x 10⁶ cells/mL for monocyte and BaF/3 chemotaxis) in Assay Medium. Add 100 µL to upper wells of chamber. Avoid forming bubbles at the level of the filter. Incubate for 3 hours at 37 °C.
5. Disassemble chamber and remove filter. Scrape cells that did not migrate from the top of the filter. Stain the filter with the Hema 3 Staining System per manufacturer's instructions. Read O.D. of stained well filters (the 96-well filter will snap into place) per manufacturer's instructions.
6. Carefully mix media remaining in lower chemotaxis chamber and transfer to a new 96-well plate.
1. If using MTT to develop the chemotaxis assay, add 20 µL of MTT (at 5 mg/mL) to transferred cells. Incubate for 4 hours at 37 °C. Add 100 µL of Solubilization Buffer to the wells, incubate for 4 hours and read on a plate reader at 540/690 nm. Graph using 4-parameter fit equation.
2. If using Alamar Blue, add 10 µL of Alamar Blue (100 µg/mL) to transferred cells. Incubate for at least 4 hours (may incubate overnight) at 37 °C. Read plate on a fluorescent plate reader, excitation at 544 nm and emission at 590 nm. Graph using 4-parameter fit equation.
   Lymphocytes do not adhere to the underside of the filter and will migrate through to the well below. Extreme care must be taken when disassembling the chamber to avoid variations in lower chamber well volumes. Staining of the filter will ensure that cells did not adhere.

Fig. 1. Human MCP-1 chemoattracts human monocytes, exhibiting a bell-shaped dose response curve. The ED50 for this effect is typically 0.005 - 0.02 µg/mL.

Fig. 2. To measure the ability of the antibody to neutralize the chemoattractant activity of rhMCP-1 for human monocytes, rhMCP-1 was incubated with various concentrations of the antibody for 30 minutes at room temperature in a 96-well microplate. Following this preincubation period, 35 µL of the cytokine-antibody solution (containing rhMCP-1 at a final concentration of 0.1 µg/mL and antibody at the concentrations indicated) was transferred to the lower compartment of a 96-well chemotaxis chamber (NeuroProbe, Cabin John, MD). The chemotaxis chamber was then assembled using a PVP-free polycarbonate filter (8 micron pore size) and 1 x 106 cells/well was added to the top chamber. After incubation for 75 minutes at 37 °C in a 5% CO2 humidified incubator, the chamber was disassembled and the filter was fixed and stained using Leukostat (Fisher Scientific). The optical density of the filter, which is proportional to the number of cells that migrated across the filter, was then read in a microplate reader set at 540 nm. As shown in figure 2, the ND50 for this lot of antibody is approximately 0.8 - 2.5 µg/mL.

Troubleshooting

1. Using glass will activate platelets and cause the release of factors affecting WBC integrity and release. Using glass will also result in the release of chemokines that will affect chemotaxis.
2. Ensure that the chemotaxis chamber is clean and dry.
3. Bubbles in either the lower or upper parts of the chamber will inhibit migration.
4. Disassemble the chamber carefully so that the volume in the lower chamber remains uniform.
5. Failure to adequately remove all cells that have not migrated from the top of the filter will cause abnormal staining.
   

References