Migration assays were conducted using the ChemoTx system (Neuro Probe, Inc., Gaithersburg, MD). Splenocytes isolated from mice inoculated with WT or Hsp70−/−E1A/Ras MEF transformants 5 days prior, were introduced into the upper chamber above WT or Hsp70−/− MEF transformants and after a 4 hour incubation at 37°C, the cell number in the lower well was enumerated using a Scepter cell counter (EMD Millipore, Billerica, MA).
Chemotx system
Manufactured by Neuro Probe
Sourced in United States
The ChemoTx System is a chemotaxis and cell migration assay platform. It is designed to allow the user to measure the directional movement of cells in response to a chemical gradient. The system consists of a chemotaxis chamber and accompanying software.
Automatically generated - may contain errors
Lab products found in correlation
Celltiter glo luminescent cell viability assay, by Promega (3 mentions)
Ccl21, by Thermo Fisher Scientific (2 mentions)
Ccl19, by Thermo Fisher Scientific (2 mentions)
Apotome, by Zeiss (2 mentions)
Scepter cell counter, by Merck Group (1 mentions)
Ficoll hypaque, by Merck Group (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Biotek microtiter plate reader, by Agilent Technologies (1 mentions)
Pkh26, by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Amd3100, by Merck Group (1 mentions)
Macsxpress isolation kit, by Miltenyi Biotec (1 mentions)
Bmg polarstar, by BMG Labtech (1 mentions)
Imagej, by Leica camera (1 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions)
Trypan blue solution, by Thermo Fisher Scientific (1 mentions)
Diff quik, by Sysmex (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
18 protocols using chemotx system
1
Splenocyte Migration Assay for Transformed MEFs
2
Canine Monocyte Migration Assay
3
Renal Fibroblast Migration Assay
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Migration of renal fibroblasts was determined after 4 hours of incubation with 10 μM LPC with or without recombinant ATX using the ChemoTX system (Neuro probe, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Briefly, after media containing stimuli was added to each well of the 96-well plate, a fibronectin-coated membrane with 8 μm pore size was placed on top of the plate such that the media made contact with the bottom side of the membrane. Then, renal fibroblasts were put on the top-side of membrane. Following a 4-hour incubation, the top-side of membrane was scraped with a cotton swab, and the number of renal fibroblasts that migrated to the bottom-side of membrane were counted. A migration index was determined by the ratio of the number of migrated cells in the presence of stimuli to that in control media containing LPC alone. Renal fibroblasts were pre-treated with PAT-048 or AM152 for 30 min prior to the addition of the ATX and LPC.
4
Chemotaxis Assay with Antimicrobial Peptide
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Chemotaxis assays were performed using the ChemoTx System from NeuroProbe with modification (NeuroProbe, Gaithersburg, MD). Briefly, samples of rhBD-2 were prepared at 3 concentrations (100 ng/μl, 200 ng/μl and 400 ng/μl) selected following kinetic analysis of optimum chemotaxis for CEM-SS cells, then incubated for 72 hr at 37°C in either 10μM MGO or 0.0067M PBS, pH 7.5. CEM-SS cells were initially grown in RPMI media, then resuspended in “chemotaxis media” (serum-free HG-DMEM with 1% BSA). The chemotactic mix containing rhBD-2 at final concentrations of 0, 10, 20 and 40 μg/ml, with or without MGO, or 10 nM stromal cell-derived factor 1 (SDF-1) as positive control (shown previously to induce a chemotactic response in CEM-SS cells, unpublished) were added to lower wells (30 μl) of a ChemoTx 96-well chamber. CEM-SS cell suspension was loaded into top wells of the chamber for a final cell concentration of 1 x 106 cells/ml, and the chamber incubated for 2 hr in 5% CO2 at 37°C. After incubation CyQuant cell dye (Life Technologies, Grand Island, NY) was injected into the lower sample wells, and developing fluorescence quantitated using a BioTek microtiter plate reader (BioTek, Winooski, VT).
5
Tumor-Derived Conditioned Medium Effects on Stem Cell Migration
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The effects of tumor-derived conditioned medium on stem-cell migration were assessed using a transwell migration assay (ChemoTx® System, Neuro Probe Inc.). Passage 3 hADSCs (1 x 104 cells in 33 μL medium) pre-labeled with PKH26 (Sigma-Aldrich) were loaded into each transwell. Tumor-derived conditioned medium (hypoxia or normoxia) and base medium (55 μL/well) were loaded into a 96-well plate, into which the transwell was inserted. After 4 h of incubation, cells in the upper chamber were removed via aspiration and the porous membrane was carefully removed. Non-migrating cells on the origin side of the membrane were removed by gently wiping with a cotton swab. The nuclei of the cells on the membrane were counterstained with 10 mM Hoechst 33342 (Sigma-Aldrich) for 10 min at room temperature. At least five random fields of view on each membrane were selected, and cells were imaged with a fluorescence microscope (Carl Zeiss, ApoTome). Automated counting of nuclei was performed with ImageJ 1.5i (NIH). To determine whether the CXCR4 antagonist AMD3100 hindered hADSC migration, hADSCs were pre-incubated with 100 ng/mL AMD3100 (Sigma-Aldrich) for 1 h before they were placed in the upper compartment of the transwell.
6
Heparin Impact on Platelet Chemokine Secretion
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To characterize the impact of heparin on platelets´ chemokine release, a boyden chamber transmigration approach with 3 μm pores was applied. Neutrophils were isolated from EDTA blood using a MACSxpress isolation kit according to manufacturer´s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Neutrophils (2 × 105 in 50 μL RPMI medium containing no FCS) were pipetted on the upper part of the membrane of the chemotaxis transmigration chamber (ChemoTx System, Neuro Probe, Gaithersburg, USA). In the bottom of the chamber, platelet releasates (from platelets treated with heparin and activators, respectively) were added and the transmigration plate was incubated for 2 h at 37°C. Afterwards, transmigration plate was centrifuged in a plate centrifuge for 5 min at 350 × g. The transmigrated neutrophils in the bottom part of the chamber were transferred to a white 96-well plate and subsequently quantified by the CellTiter-Glo Luminescent Cell Viability Assay (Promega, Mannheim, Germany) using a microplate reader (BMG POLARstar, BMG Labtech).
7
Chemotaxis Assay for Monocyte Migration
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Chemotaxis of CD14+ monocytes was performed using a 96-well ChemoTX system (Neuroprobe) with membranes containing 8 μm pores per manufacturer’s protocol. Briefly, monocytes were rested overnight in serum-free RPMI-1640 then 105 cells in 80 μl were plated onto top chamber of the membrane. Lower chambers contained 325 μl of complete RPMI-1640 with or without addition of rHMGB1 (200 ng/ml), anti-HMGB1 Ab (10 μg/ml), LPS (10 μg/ml), and pooled acellular MPE fluid (50%). ChemoTX plates were incubated at 37°C, 5% CO2, for 12 h. Transwell membranes were wiped with cotton and washed in PBS to remove unbound cells, then stained with 0.2% crystal violet. Transwell membranes were imaged using a Leica DMI 3000B digital microscope and cell quantitation performed by ImageJ (United States National Institutes of Health) to extrapolate cell counts of three randomly selected fields to represent the total area of the membrane well.
8
Chemotaxis assay of S1P-induced cell migration
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Cells were grown to reach 60–70% confluence, and then starved overnight in DMEM containing 0.1% BSA. The ChemoTx System was used for chemotaxis assay (NeuroProbe, Gaithersburg, MD). Briefly, cells were washed twice with PBS, and then incubated for 30 min at 37 °C in the presence of 1 μM calcein-AM. Stained cells were washed three times with PBS, and detached with trypsin. Approximately 8000 cells were placed directly onto chemotaxis filters. The bottom chambers were filled with DMEM containing different concentrations of S1P (0.01, 0.1, or 1 µM). Cells were incubated for 6 h at 37 °C. The non-migratory cells on the top of the filter were removed by gently wiping the filter with a cotton swab. Fluorescence of migrated cells into the bottom chamber was measured in a multi-well fluorescent plate reader (Fluoroskan Ascent FL, ThermoElectron Corporation, Excitation, 485 nm; Emission, 538 nm). Fluorescence was converted to numbers of cells based on a standard curve, generated by seeding known numbers of fHASCs at the bottom of the chamber.
9
Dendritic Cell Migration Assay
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Supernatant derived from uninfected, or neutrophils cultured under different treatment conditions were placed in the lower compartment of a transwell plate (96 well plate, 3.2mm diameter 5um pore size, ChemoTx System, NeuroProbe, UK). 105 DCs were placed on top of the filter. Migration in medium alone acts as the negative control. After 3h of incubation at 37°C, 7 mM EDTA [prepared from 0.5M EDTA (Thermo Fisher Scientific, Cat: PR-V4231)] was added to the bottom wells for 10 min to release adhered cells from the well and filter. Cells from the lower chambers were then stained with trypan blue solution (Thermo Fisher Scientific, Cat: 15250061) and counted on a hemocytometer.
10
Monocyte Chemotaxis Assay Under Normoxia and Hypoxia
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A chemotaxis assay was performed with 96‐well ChemoTx System (Neuro Probe Inc. Gaithersburg, MD, USA) using isolated PMs in accordance with the manufacturer's instruction. PMs (2 × 106 cells/mL) in DMEM containing 0.1% BSA were loaded into the upper wells. The lower wells separated by polycarbonate membrane with 8‐μm pores were filled with the same medium containing monocyte chemotactic protein‐1 (MCP‐1) (0, 10, 25 ng/mL). After incubation for 16 h at 37°C under normoxic or hypoxic conditions, the number of migrated cells per field on the lower surface of the membrane was counted after staining with Diff‐Quik (Sysmex, Kobe, Japan). Experiments were performed in duplicate and repeated at least three times (Ikeda et al. 2013 ).
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