For Boyden chamber assays, QCM Chemotaxis Cell Migration Assay (Sigma-Aldrich) was used according to the manufacturer’s instructions. Scratch assays were conducted in 6-well plates. Scratches were inflicted in each well using a micropipette tip, and cells were washed once before adding compounds. At least three images per scratch were used for analysis using TScratch (https://www.cse-lab.ethz.ch/software/ ). In all migration experiments, cells were starved for 48 h prior to assay start in order to prevent proliferation
Qcm chemotaxis cell migration assay
Manufactured by Merck Group
Sourced in Germany
The QCM Chemotaxis Cell Migration Assay is a laboratory equipment used to measure cell migration in response to chemical stimuli. It provides a quantitative assessment of the migratory capacity of cells under different experimental conditions.
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Lab products found in correlation
Biocoat growth factor reduced matrigel invasion chamber assay, by Corning (2 mentions)
Cell proliferation elisa brdu kit, by Merck Group (2 mentions)
Cell death detection elisaplus kit, by Merck Group (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Sdf 1, by Thermo Fisher Scientific (1 mentions)
Pdgf bb, by Thermo Fisher Scientific (1 mentions)
Pdgf aa, by Thermo Fisher Scientific (1 mentions)
Incucyte live cell analysis system, by Sartorius (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
14 protocols using qcm chemotaxis cell migration assay
1
Evaluating Cell Migration Capacity
2
Scaffold-Induced hBMSC Migration Assay
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Given the substantial ion release from the cell-loaded scaffolds at the first 3 culture days, they were incubated for 3 days in a growth medium and then the condition media (CM) were harvested for hBMSC migration studies. The latter were performed using colorimetric QCM Chemotaxis Cell Migration Assay (Sigma-Aldrich). Before cell seeding into the chambers (24-well plates), they were cultured in a serum free medium for 24 h in order to inhibit cell proliferation. HBMSC were then seeded in the upper chamber (8 μm pore diameter) at the density of 1.0 × 106 cells/mL and exposed to condition media (CM) that had been harvested from the studied scaffolds and transferred to the lower chambers. Cells were incubated for 24 h at 37 °C. The control in this experiment was the growth medium that had been used to incubate the scaffolds for 3 days, but without any contact with the scaffolds. Cells that had migrated through the membranes were stained according to the manufacturer’s protocol, and the number of migrated cells was determined quantitatively by reading absorbance at 650 nm.
3
Rat and Human MSC Isolation, Characterization and Migration
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Rat MSC were isolated aseptically from bone marrow derived from the femurs of 6 to 10 week old male Wistar rats and characterized by flow cytometry analysis for selected surface markers per protocol as previously described [10 (link), 11 (link)]. rMSC cell migration was assessed using the QCM Chemotaxis Cell Migration Assay (8 μM, EMD Millipore, Billerica, MA) per manufacturer’s instructions. Monocyte chemoattractant protein-1 (MCP-1), stromal cell-derived factor 1 (SDF-1), platelet-derived growth factor-AA (PDGF-AA), and platelet-derived growth factor-BB (PDGF-BB) used to assess cell migration was obtained from Peprotech (Rocky Hill, NJ). rMSC at passage 2 to 4 were administered for transplantation.
Human MSC were derived and characterized by the NIH Bone Marrow Stromal Cell Transplantation Center [12 (link), 13 (link)] and cultured as previously described [14 (link)]. Superparamagnetic iron oxide nanoparticles (SPION) (ferumoxides (Feridex, Bayer AG, Germany) were complexed to the clinical transfection agent protamine sulfate as previously described [15 (link)]. hMSC at passage number less than 5 were magnetically labeled by co-culture with ferumoxides-protamine sulfate (Fe-Pro) in fresh media for 2 hours at 37°C as described [16 (link)].
Human MSC were derived and characterized by the NIH Bone Marrow Stromal Cell Transplantation Center [12 (link), 13 (link)] and cultured as previously described [14 (link)]. Superparamagnetic iron oxide nanoparticles (SPION) (ferumoxides (Feridex, Bayer AG, Germany) were complexed to the clinical transfection agent protamine sulfate as previously described [15 (link)]. hMSC at passage number less than 5 were magnetically labeled by co-culture with ferumoxides-protamine sulfate (Fe-Pro) in fresh media for 2 hours at 37°C as described [16 (link)].
4
Comprehensive Cell Assays for Drug Screening
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For proliferation studies, cells were plated at ~30% confluence and images obtained every 2 hours for 72 hours. An IncuCyte® Live-Cell Analysis System (Essen Biosciences) collected images and proprietary IncuCyte® software calculated confluence at each time point.
Migration assays were performed with the QCM Chemotaxis Cell Migration Assay (EMD Millipore). Cells were treated with DOX or saline and after 24 hours transferred to a Boyden chamber membrane (8 μm pore) in serum-free media. The lower chamber was filled with 10% FBS media. After 24 hours, migrated cells were stained and the absorbance measured. (EMD Millipore).
Cell cycle arrest was assessed by propidium iodide (PI) DNA-staining. Three hours after treatment, cells were detached, and a single cell suspension was generated. Fluorescence was quantified by the UTHSCSA Flow Cytometry Core Center.
Migration assays were performed with the QCM Chemotaxis Cell Migration Assay (EMD Millipore). Cells were treated with DOX or saline and after 24 hours transferred to a Boyden chamber membrane (8 μm pore) in serum-free media. The lower chamber was filled with 10% FBS media. After 24 hours, migrated cells were stained and the absorbance measured. (EMD Millipore).
Cell cycle arrest was assessed by propidium iodide (PI) DNA-staining. Three hours after treatment, cells were detached, and a single cell suspension was generated. Fluorescence was quantified by the UTHSCSA Flow Cytometry Core Center.
5
Quantitative Cell Migration Assay
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Cell migration was measured with the use of QCM Chemotaxis Cell Migration Assay (Merck) based on the Boyden chamber principle. Cells were detached from the wells and suspended in chemoattractant-free media. The cell suspension was then added to inserts (8um, PC membrane) and medium containing 10% FBS to the lower chamber. After incubation, inserts were transferred into clean wells containing Cell Stain. Cotton-tipped swabs were used to remove the non-migratory cells layer from the interior of the inserts. Then inserts were transferred to clean wells containing Extraction Buffer. After incubation, absorbance was read at 570 nm on a microplate reader (iMark, Bio-Rad).
6
Quantifying GBM Cell Migration and Invasion
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The cell migration rates of control and PELP1 silenced GBM cells were determined using a colorimetric QCM chemotaxis cell migration assay (EMD Millipore, Billerica, MA) as per the manufacturer’s instructions. The invasive potential of control and PELP1 knockdown cells were determined using Corning® BioCoat™ Growth Factor Reduced Matrigel Invasion Chamber assay according to the manufacturer’s instructions.
7
NLRP3 Inflammasome Migration Assay
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Migration was analyzed in Boyden chambers (HCASMC: 8 µm pore size) using QCM Chemotaxis Cell Migration Assay (Merck). Inserts were coated with sterile-filtered 10 µg/ml Typ 1 collagen (Corning Collagen Typ I rat) and 0.2 × 106 cells were seeded on the insert in serum-starved media with 0.2% FCS (SFM). The well was filled with serum-containing media (10% FCS). PDGF (10 ng/ml, Peprotech) was used as positive control, Actinomycin D (5 µg/ml, Merck) as negative control. Extracellular NLRP3-YFP inflammasome particles were added to the cells (3:1 particles/ cell) in the insert for 4 h at 37 °C. Then, migrated cells of the lower side of the membrane were stained with cell stain crystal violet and remaining cells in the insert were removed with a Q-tip. After washing, stained cells on the apical side of the porous membrane were lysed with extraction buffer and the absorbance was measured at 560 nm in a 96 well plate.
8
Evaluating Microvascular Cell Responses to Extracellular Vesicles
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HRPs and HRECs were exposed for 24 h to the four types of EVs isolated from microglial cultures, as previously described. Other microvascular cells were cultured in parallel for 24 h in the presence of the M1 cocktail, with or without the addition of thiamine.
Proliferation was assessed using the Cell Proliferation ELISA BrdU kit (Merck), and apoptosis was assessed using the Cell Death Detection ELISAPLUS kit (Merck), according to the manufacturer’s instructions.
Cell migration was measured using the colorimetric QCM Chemotaxis Cell Migration Assay (Merck): cells were seeded inside 8 µm pore polycarbonate membranes and exposed to EVs for 24 h. Subsequently, cells still inside the insert were mechanically removed and those that migrated through the membrane were stained. The dye was then extracted, and colorimetric reading was performed spectrophotometrically at 560 nm.
ROS production was assessed by exposing cells in 96 well/plates for 45 min at 37 °C to 25 µmol/L H2DCFDA (Invitrogen–ThermoFisher, Waltham, MA, USA) in medium without red phenol and FCS. Wells were then washed and fresh medium was added. Fluorescence was measured at 490 nm excitation/520 nm emission at different time points.
Proliferation was assessed using the Cell Proliferation ELISA BrdU kit (Merck), and apoptosis was assessed using the Cell Death Detection ELISAPLUS kit (Merck), according to the manufacturer’s instructions.
Cell migration was measured using the colorimetric QCM Chemotaxis Cell Migration Assay (Merck): cells were seeded inside 8 µm pore polycarbonate membranes and exposed to EVs for 24 h. Subsequently, cells still inside the insert were mechanically removed and those that migrated through the membrane were stained. The dye was then extracted, and colorimetric reading was performed spectrophotometrically at 560 nm.
ROS production was assessed by exposing cells in 96 well/plates for 45 min at 37 °C to 25 µmol/L H2DCFDA (Invitrogen–ThermoFisher, Waltham, MA, USA) in medium without red phenol and FCS. Wells were then washed and fresh medium was added. Fluorescence was measured at 490 nm excitation/520 nm emission at different time points.
9
S-equol and Liq Effects on Cell Migration and Invasion
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The cell migration and invasion were determined by using colorimetric QCM chemotaxis cell migration assay (EMD Millipore, Billerica MA) and the Corning® BioCoat™ Growth Factor Reduced Matrigel Invasion Chamber assay, respectively. ES2 and SKOV3 cells were pretreated with S-equol (100 μM) or Liq (100 μM) for 24 h and subjected to cell migration and invasion assays according to manufacturer protocols.
10
Neutrophil Chemotaxis Migration Assay
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