To determine the CXCR4-SDF1α (Stromal Derived Factor 1- alpha) interaction, chemotaxis assay was performed using Costar 24-well Transwell plates (6.5 mm diameter inserts, 8.0 µm PET membrane, Corning, USA). 600 µl of IMDM containing SDF1α (100 ng/ml, Miltenyi Biotec) was poured into the bottom chamber [29 (link)]. Linneg cells either treated with NaHS (300 µM) or with vehicle and were cultured in IMDM+10% HI-FBS for 24 h allowing for the up-regulation of CXCR4 expression. Additionally, 30 m prior to this assay replicate cells were treated with a CXCR4 antagonist, AMD3100 (10 µM) to block the receptor. Thereafter, the cells were washed and resuspended in IMDM+0.5% BSA. The cells were volumetrically enumerated and 100 µl of cell suspension (2x105 cells) was added to the top chamber of the trans well plate. The plates were then incubated for 4 h at 37°C, 5% CO2 and 95% humidity. The cells that migrated to the bottom chamber were stained with surface marker antibodies and enumerated by flow cytometry.
Costar 24 well transwell plates
Manufactured by Corning
Sourced in United States
The Costar 24-well transwell plates are a laboratory equipment product designed for cell culture applications. These plates feature a permeable membrane insert that allows for the study of cell migration, invasion, and permeability. The product provides a convenient and standardized platform for these types of experiments.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Amd3100, by Merck Group (2 mentions)
8.0 m pet membrane, by Corning (1 mentions)
Egm 2 media, by Lonza (1 mentions)
Diff quik stain set, by Siemens (1 mentions)
Egm 2, by R&D Systems (1 mentions)
Matrigel invasion chamber, by Thermo Fisher Scientific (1 mentions)
Nvp bhg712, by Merck Group (1 mentions)
Eclipse ti inverted microscope system, by Nikon (1 mentions)
5 μm pore filter, by Corning (1 mentions)
5 protocols using costar 24 well transwell plates
1
CXCR4-SDF1α Chemotaxis Assay
2
Transwell Migration and Invasion Assay
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Migration assays were performed using Costar 24-well Transwell plates (Corning, Tewskbury, MA). AVMECs (passages 6–10) and HBMVECs (passages 9–10) were plated at 100,000 cells/well in 100 μL of EGM2 media (Lonza, Walkersville, MD) with 0.1% serum in the upper chamber. In the lower chamber, either EGM2 with 0.1% serum (negative control), EGM2 full serum media (positive control), or EGM2 with 0.1% serum supplemented with EphrinB2-FC recombinant 250 ng/mL (R&D Cat. No. 7397-EB-050, Waltham, MA) or 100 nM EphB4 inhibitor19 (link) (Sigma, NVP BHG 712, St. Louis, MO) were added to the lower chamber to act as a chemoattractant. After 24 h, cells remaining in the upper chamber were aspirated; cells in the bottom chamber were fixed, permeabilized, and stained using Diff-Quik Stain Set (Siemens, Malvern, PA) as previously published20 (link),21 (link). Invasion assays were performed in a similar fashion as above using Matrigel invasion chambers (Thermo Fisher Scientific, Waltham, MA). Experiments were performed in duplicate for each cell line, with a total of ten high-power fields analyzed per cell line. Representative images were captured at ×100, and the number of migrated (or invaded) cells was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
3
Macrophage Migration Assay with GSK'074
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Bone marrow-derived macrophages were starved in 0.5% fetal bovine serum in DMEM for 24 hours, then pretreated with 100 nmol/L GSK’074 or DMSO for 2 hours. Then, 2 × 104 cells were seeded into the upper chamber of Costar 24-well transwell plates with 5-μm pore filters (Corning Inc, Corning, NY). Medium containing 100 ng/mL MCP1 and 100 nmol/L GSK’074 or DMSO was added into the lower wells. After 16 hours, cells that migrated to the bottom of the membrane were fixed, stained with 4',6-diamidino-2-phenylindole, and counted under a fluorescence microscope (Nikon Eclipse Ti inverted microscope system).
4
Chemotaxis Assay for Human CB HSCs
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Chemotaxis assays were performed by using Costar 24-well transwell plates with 6.5 mm diameter inserts with 5.0 μm pores (Corning, Inc., Corning, NY, USA). 650 μL of pre-warmed IMDM medium (37 °C) that contained 0.5% bovine serum albumin (Sigma-Aldrich) and SDF-1 (0, 25, 50 ng/mL) was added to the bottom well. Cells were suspended at 1 × 105 cells/100 μL in IMDM medium and loaded to the upper chamber of the transwell. Transwell plates were placed in a 37 °C incubator with 95% humidity and 5% CO2for 4 h. Percent migration was measured using flow cytometry with number of cells in the bottom chamber divided by number of cells placed in the upper chamber. To calculate percent migration of CB HSCs, phenotypic HSCs(CD34+CD38−CD45RA−CD90+CD49f+ cells) was determined by surface staining and flow cytometry analysis. Human CB HSC chemotaxis was calculated as number of HSCs in the bottom chamber divided by number of HSCs loaded in the upper chamber. For AMD3100 administration, cells were treated with 5 μg/mL AMD3100 (239820, Sigma-Aldrich) for 30 min right before the chemotaxis assay.
5
Chemotaxis Assay of Cord Blood HSCs
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Chemotaxis assays were performed in Costar 24-well transwell plates with 6.5 mm diameter inserts with 5.0 μm pores (Corning, NY, USA). Briefly, 650 μl of IMDM medium (37 °C) that contained 0.5% bovine serum albumin (Sigma-Aldrich) and SDF-1 (0, 50 ng/ml) was added to the bottom well. Cells were suspended at 1 × 105 cells/100 μl in IMDM medium and loaded to the upper chamber of the transwell. Transwell plates were placed in a 37 °C incubator with 95% humidity and 5% CO2 for 4 h. Percent migration was measured using flow cytometry with number of cells in the bottom chamber divided by number of cells placed in the upper chamber. To calculate the percent migration of CB HSCs, phenotypic HSCs was determined by surface staining and flow cytometry analysis. Human CB HSC chemotaxis was calculated as number of HSCs in the bottom chamber divided by number of HSCs loaded in the upper chamber. For AMD3100 administration, cells were treated with 5 μg/ml AMD3100 (239820, Sigma- Aldrich) for 30 min right before the chemotaxis assay.
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