To assay for cell migration, cells were FACS-isolated and seeded onto the ChemoTx Chemotaxis System (106-8, NeuroProbe). Specifically, one side of the Transwell filter was coated with fibronectin (Biomedical Technologies Inc) for 30 min followed by two PBS washes. Wells were filled with DMEM + 10% FBS. After the filter layer was carefully placed such that the fibronectin-coated side was face down into the well, 5,000 sorted cells (resuspended in DMEM + 10% FBS) were seeded on top of the membrane on the noncoated side. Chemotaxis plates were placed in 5% CO2 at 37 °C for 12 h. Filter membranes were then fixed, stained and mounted onto slides. Quantifications represent the averages of six individual assays.
Chemotx chemotaxis system
Manufactured by Neuro Probe
Sourced in United States
The ChemoTx chemotaxis system is a tool used for conducting cell migration experiments. It provides a platform for researchers to study the directional movement of cells in response to chemical gradients. The system consists of a multi-well plate design with a porous membrane, allowing for the controlled introduction of chemoattractants and the observation of cell migration across the membrane.
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Lab products found in correlation
Fibronectin, by Biomedical Technologies (1 mentions)
Cd16 32 antibody, by BioLegend (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Triad series multimode detector, by Dynex (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by Tree Star (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Murine cxcl9, by Thermo Fisher Scientific (1 mentions)
8 protocols using chemotx chemotaxis system
1
Cell Migration Assay with Transwell
2
Chemotaxis Assay for Mouse Neutrophils
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In vitro chemotaxis assays were done as previously described73 (link). Mouse blood was collected by cardiac puncture and red blood cells were lysed for 5 min in lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA). The leukocytes were then washed in FACS buffer, blocked with CD16/32 antibody (BioLegend), and stained with fluorophore-conjugated primary antibodies (anti-mouse CD11b and Ly6G). Different recombinant chemokines (rCXCL4, rCXCL5, and rCXCL7; R&D cat. no. 595-P4, 433-MC, and 1091-CK) were added as chemoattractants at 2 µg/mL into the lower chamber of a ChemoTx chemotaxis system (Transwell filter with 5-µm pore size; Neuroprobe). Stained leukocytes were plated in the upper chamber. Both the upper and lower chambers contained RPMI. After 2 h, the content of the lower chamber was collected and mixed with propidium iodide (for assessing cell viability) and Bright Count absolute counting beads (Invitrogen). Samples were then analyzed by FACS to determine the number of migrated CD11b+Ly6G+ neutrophils.
3
Quantitative Cell Migration Assay
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Approximately 1 × 106 cells/mL were suspended in MEM media supplemented with 0.1% (v/v) BSA and incubated with 1 μg/mL of fluorogenic calcein-AM (Invitrogen) for 30 min at room temperature in the dark. Cells were then centrifuged at 1,500 rpm for 5 min, washed three times with MEM supplemented with 0.1% (v/v) BSA, and resuspended at the density of 8 × 105 cells/mL with MEM containing 0.1% (v/v) BSA. Afterwards, 4 × 104 cells were suspended in 49 mL of MEM before placing onto the filter (12-µm pore size) of 96-well ChemoTx® chemotaxis system (Neuro Probe), which was pre-coated with Geltrex (Life Technologies). Cells were allowed to migrate from the filter to the bottom compartment containing 29 µL of MEM supplemented with 10% (v/v) FBS at 37°C for 6 h. The non-migrated cells on the top of the filter were gently removed with a paper towel before the fluorescence intensity generated from the calcein-labeled cells in the bottom compartment was measured with excitation and emission wavelength at 485 and 520 nm, respectively, in a Triad series multimode detector (Dynex Technologies).
4
Quantifying Cell Migration Using Chemotaxis Assay
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Cells were seeded on the membranes of the 96-well plate ChemoTx® chemotaxis system (116-8; Neuro Probe, Inc., Gaithersburg, MD, USA) in a culture medium containing the growth factors as indicated for treatment. After 24 hours, the membrane was washed twice in phosphate-buffered saline and fixed with 70% ethanol. The nonmigrated cells were removed by cotton swab from the upper side of the membrane. The membrane was stained with 0.5% crystal violet, and subsequently visualized and quantified by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).16 (link)
5
Chemotaxis Assay for Murine and Human CD8+ T cells
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Murine CD8+ T cells stimulated with anti-CD3 and anti-CD28 for 24 hr and rested for 2 days in a complete RPMI culture medium containing 10% FBS, 1% Glutamine, 1× Pen/Strep, and 10 ng/ml IL-2 (Invitrogen). Human CD8+ T cells were stimulated with anti-CD3 and anti-CD28 for 24 hr. The chemotaxis assay was performed using 96-well ChemoTx chemotaxis system with 3 μm pore size (Neuro Probe) according to manufacturer’s protocol. Briefly, the bottom wells were filled with 30 μl of migration buffer with or without 100 ng/ml murine or human CXCL9 or CXCL10 (PEPROTECH). To coat the 3 μm pore, fibronectin (30 μg/ml, Sigma) and sEVs (30 ug/ml) were mixed at 1:1 ratio and dropped onto the filter top for 24 hr at 4 °C. CD8+ T cells were applied to the top of filters rinsed with PBS. After 3-6 hr incubation at 37 °C, migrated cells collected from the bottom wells were quantified using a cell counter.
6
Chemotaxis Assay for Macaque Bone Marrow
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The 96-well ChemoTx chemotaxis system (NeuroProbe Inc., 5um pore) was used for chemotaxis assays. The lower wells were blocked with 301 μl of 1% BSA for 30 min at room temperature, which was aspirated and replaced with 301 μl of plasma from pre- or post-infected macaques as test samples. 301 μl of RPMI-1640 /0.1% BSA buffer was included in the experiments as buffer controls. Single cell suspensions from bone marrow of naive macaques were collected and 2 x 105 bone marrow single cell suspensions were re-suspended in 50 μl RPMI-1640 /0.1% BSA and loaded above the membrane. After incubation for 2 hr at 37°C, 5% CO2, the top wells were removed with a scraper and the migrated cells in the bottom wells were counted, and stained with the antibody mixture: CD3-PE-Cy7, CD14-V450, HLA-DR-APC-Cy7, Lin-FITC, CD33-PE, and CD11b-PE-Cy5, Cd45-Alexa 700, and yellow viability dye. 50 μl of CountBright absolute counting beads (Molecular Probes) were added to each tube before data acquisition using an LSRII flow cytometer, and FlowJo software (Tree Star Inc.) was used for data analyses. Chemotaxis index was defined as the ratio of the absolute cell numbers in the test samples over the absolute cell numbers in the buffer control samples.
7
Neutrophil Migration Assay
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5 × 104 neutrophils were plated on a semipermeable membrane (ChemoTx Chemotaxis System, pore size 5 μm, Neuroprobe) in the presence or absence of KC at T0 and following 6 hours of aging, incubated for 1 hour at 37°C, spun at 300 g for 10 minutes, and hemocytometer counts performed. The number of cells in each well was expressed as a percentage of the total number of cells loaded into each well.
8
CAR T Cell Transmigration Assay
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