A 96-well chemotaxis chamber (Neuro Probe, Inc., Gaithersburg, MD) was used to measure neutrophil chemotaxis, as previously described [18] (link). Briefly, the lower wells of a microplate were loaded with 30 μl of control medium (RPMI) containing different concentrations of IL-8 (10− 10–10− 7 M), a potent CXC chemokine that is used to induce human neutrophil chemotaxis in vitro. To assess the chemotactic response of neutrophils to adenosine, 30 μl of adenosine (10− 8–10− 5 M) was added to the lower wells. Assays were performed in duplicate. 30 μl of neutrophils (2 × 106 ml− 1) ± adenosine ± inhibitors ± erythrocytes was placed directly onto the 5 μm filter membrane. The chemotaxis chamber was incubated for 30 min (37 °C, 5% CO2) as described previously [19] (link). The number of cells that had migrated into the lower chamber was calculated as a percentage of the total number of cells added to the filter.
96 well chemotaxis chamber
Manufactured by Neuro Probe
Sourced in United States
The 96-well chemotaxis chamber is a laboratory equipment designed to measure cell migration in response to chemical stimuli. It consists of a multi-well plate with a semi-permeable membrane separating the upper and lower chambers. Cells are seeded in the upper chamber, and chemical agents are added to the lower chamber, allowing for the observation and quantification of cell migration through the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Chemotaxis chamber, by Neuro Probe (2 mentions)
Polycarbonate membrane, by Neuro Probe (2 mentions)
Fibronectin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hetasep, by STEMCELL (1 mentions)
Fluoroskan ascent microplate reader, by Thermo Fisher Scientific (1 mentions)
Dld 1, by Korean Cell Line Bank (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Sw620, by Korean Cell Line Bank (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
8 protocols using 96 well chemotaxis chamber
1
Neutrophil Chemotaxis Assay with Adenosine
2
Neutrophil Chemotaxis Measurement Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neutrophil chemotaxis was measured in a 96-well chemotaxis chamber (Neuroprobe Inc., Gaithersburg, MD, USA) using the method of Frevert et al. [23 (link)] with modifications. Wells were filled with fMLP (50 nM), RPMI 1640 medium, or neutrophils (5 × 104) resuspended in RPMI 1640 medium. A filter membrane was positioned over the loaded wells and 25 μL neutrophils (2 × 106/mL) was placed directly onto 3.0-μm filter sites. The chamber was incubated under 5% CO2 at 37 °C for 1 h. Unmigrated neutrophils were removed from the upper surface of the filter by wiping and washing with 25-μL aliquots of RPMI 1640 medium. Neutrophils that migrated to the underside of the filter and into the lower wells were counted with a hemocytometer. To dislodge any migrated cells adherent to the underside of the filter membrane, the plate and attached filter were centrifuged at 350 × g for 10 min. The filter was removed and the neutrophils in the wells of the chemotaxis plate were resuspended and counted with a hemocytometer.
3
Transwell Assay for SMC Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 96-well chemo tax is chamber (Neuro Probe, Inc., Gaithersburg, MD, USA) was used for the trans well assays. The polyvinyl membrane, containing 8 µm pores, was coated with 10 µg/ml fibronectin (Sigma-Aldrich; Merck KGaA) for 2 h, and then air-dried. The lower chamber was filled with DMEM containing 0.25% bovine serum albumin (BSA) (Sigma-Aldrich; Merck KGaA), with or without 3, 10, 30 or 100 ng/ml TIMP-3. SMCs were then seeded onto the filter at 2×105 cells per well, in DMEM containing 0.25% BSA. The plates were incubated for 5 h in a humidified chamber at 37°C. The chambers were disassembled and the membranes were fixed in methanol and stained with hematoxylin and eosin. Cell nuclei were counted across ten high power fields per membrane, using a light microscope. Results were presented as the mean number of cells migrated per field.
4
Neutrophil Chemotaxis and Adhesion Assay
5
Leukocyte Chemotaxis Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total leukocytes were isolated from whole blood (collected from either humans or mice, Section 2.3 ) using HetaSep (Stemcell Technologies Canada Inc., BC, Canada) and following the manufacturer’s protocols. Leukocytes were then resuspended in Roswell Park Memorial Institute medium 1,640 (RPMI 1640) and diluted to 200,000 cells per 100 μl. Chemoattractants (50 μl) were loaded into the lower wells of a 96-well chemotaxis chamber (Neuro Probe Inc., MD, United States), and a polycarbonate filter (3 µm pores) was placed on top. Total leukocytes were loaded into the upper wells and were left to migrate towards the chemoattractant at 37°C in 5% CO2 for 30 min. Chemoattracted leukocytes were then transferred to a 96-well, black, clear-bottom plate. Measurements were taken in triplicate determination. For quantitation, Hoechst 33342 (1pM) (Thermo Fisher Scientific) was added to reach a final volume of 100 µl per well, and fluorescence was measured using a Fluoroskan Ascent microplate reader (Thermo Fisher Scientific). The number of chemoattracted leukocytes was calculated using 4 parameter logistic regression.
6
CRC Cell Line Transfection and Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CRC cell lines, DLD-1 and SW620, were obtained from the Korean Cell Line Bank (Korea) and maintained in RPMI-1640 (Gibco, U.S.), supplemented with 10% foetal bovine serum (Gibco, U.S.), at 37 °C in a 5% CO2 incubator. Cells (1.5 × 104) were seeded in triplicate in 24-well plates. After 24 h, adhered cells were transfected with empty, acceptor gene, or fusion gene vector for 48 and 72 h. To check cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, U.S.) was added to washed cells at a final concentration of 0.5 mg/mL. After 1-h incubation, absorbance was measured by spectrophotometry at 570 nm (Hewlett packard, U.S.).
After 48-h transfection of CRC cells with the appropriate vectors, migration assay was performed in triplicate using membrane filters (8 μm pore size) in disposable 96-well chemotaxis chambers (Neuro Probe; Gaithersburg, U.S.). Cells (3 × 103 and 5 × 103 of DLD-1 and SW620, respectively) were resuspended (50 μL), loaded into the upper chambers on membrane filters coated with 5 mg/mL fibronectin, and incubated for 4 h at room temperature. After 18 h, cells beneath the membrane were fixed, stained with Hoechst33342 (Sigma-Aldrich, U.S.), and counted by fluorescence microscopy at 10× magnification.
After 48-h transfection of CRC cells with the appropriate vectors, migration assay was performed in triplicate using membrane filters (8 μm pore size) in disposable 96-well chemotaxis chambers (Neuro Probe; Gaithersburg, U.S.). Cells (3 × 103 and 5 × 103 of DLD-1 and SW620, respectively) were resuspended (50 μL), loaded into the upper chambers on membrane filters coated with 5 mg/mL fibronectin, and incubated for 4 h at room temperature. After 18 h, cells beneath the membrane were fixed, stained with Hoechst33342 (Sigma-Aldrich, U.S.), and counted by fluorescence microscopy at 10× magnification.
7
Chemotaxis Quantification Assay
8
Chemotaxis Quantification Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemotaxis was assayed in 96-well chemotaxis chambers with polycarbonate membranes (pore size, 5 uM; Neuroprobe). CyQUANT dye mix was used for quantification of migrating cells. After 2 h of incubation, fluorescence was measured in a FluoroSkan Ascent fluorescent plate reader. fMLP was placed in the bottom well of a Neuroprobe chemotaxis chamber. Cells were spun (100 μl, containing on average 4 × 104 cells), then were resuspended in 50 μl DMEM and placed on top of the filter in the chemotaxis chamber. After 3 h of incubation, the number of cells migrating to the lower chamber was determined with CyQUANT. The chemotactic index was determined by division of the number of migrating cells (as determined by CyQUANT fluorescence) by the number of input cells and the results were normalized to 1 on the basis of migration of untreated cells.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!