Migration assay
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Peripheral blood samples at TP2 in EDTA tubes are diluted with PBS supplemented with 2 mM EDTA and 0.5% BSA (v/v, 1∶1); 30 mL of diluted blood solution is then loaded in a 50-mL LeucoSep tubes with 15 mL of Histopaque 1077 and centrifuged at 850×g for 25 min.
- The buffy coat layers containing white blood cells are collected and washed twice with sterile PBS.
- Cells are then resuspended in EBM-2 supplemented with 0.1% BSA.
- 0.5–1×107 cells/mL of cells are loaded onto the upper part of ThinCert™ tissue culture polystyrene inserts (452.4 mm2 culture surface, 3.0 µm pore size , , ) which are pre-assembled on 6-well plates containing medium as above with or without 100 ng/mL of recombinant human SDF-1α or β-NGF , respectively.
- Cells are then incubated at 37°C in a humidified incubator with 5% CO2 for 18 hrs.
- Cells from both parts of the transwell inserts are dissociated…
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Peripheral blood samples at TP2 in EDTA tubes are diluted with PBS supplemented with 2 mM EDTA and 0.5% BSA (v/v, 1∶1); 30 mL of diluted blood solution is then loaded in a 50-mL LeucoSep tubes with 15 mL of Histopaque 1077 and centrifuged at 850×g for 25 min.
- The buffy coat layers containing white blood cells are collected and washed twice with sterile PBS.
- Cells are then resuspended in EBM-2 supplemented with 0.1% BSA.
- 0.5–1×107 cells/mL of cells are loaded onto the upper part of ThinCert™ tissue culture polystyrene inserts (452.4 mm2 culture surface, 3.0 µm pore size , , ) which are pre-assembled on 6-well plates containing medium as above with or without 100 ng/mL of recombinant human SDF-1α or β-NGF , respectively.
- Cells are then incubated at 37°C in a humidified incubator with 5% CO2 for 18 hrs.
- Cells from both parts of the transwell inserts are dissociated with Accutase and collected into FACS tubes.
- Following washing using PBS, migrated and non-migrated cells are stained with KDR-FITC, CD133/2 (293C3)-APC, CD34 (8G12)-PE-Cy7 and CXCR4 (CD184)-PE antibodies for flow cytometry analysis as described above.
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The amount of NGF released from gels with and without the C6S-binding peptide and C6S was quantified.
- Gels (50 µl) were prepared in triplicate in 2 ml siliconized tubes, as previously described, that contained 2 µg/ml NGF (13257019).
- After the gels were cross-linked, 2 ml of 1xPBS was added onto each gel.
- At each time point, 500 µl of buffer was collected from each tube and immediately replaced with 500 µl of buffer using siliconized pipets and tubes.
- NGF release was monitored over 48 h, and samples were stored at -20°C.
- After 2 d, the gels were broken up with a spatula and digested in 10 units/ml collagenase and 0.4 units/ml chondroitinase ABC for 48 h at 37°C with gentle shaking.
- The amount of NGF in the collected samples was quantified with a human β-NGF ELISA development kit (900-K60).
- The absorbance of each well was measured at 405 nm and 650 nm after 10 min…
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The amount of NGF released from gels with and without the C6S-binding peptide and C6S was quantified.
- Gels (50 µl) were prepared in triplicate in 2 ml siliconized tubes, as previously described, that contained 2 µg/ml NGF (13257019).
- After the gels were cross-linked, 2 ml of 1xPBS was added onto each gel.
- At each time point, 500 µl of buffer was collected from each tube and immediately replaced with 500 µl of buffer using siliconized pipets and tubes.
- NGF release was monitored over 48 h, and samples were stored at -20°C.
- After 2 d, the gels were broken up with a spatula and digested in 10 units/ml collagenase and 0.4 units/ml chondroitinase ABC for 48 h at 37°C with gentle shaking.
- The amount of NGF in the collected samples was quantified with a human β-NGF ELISA development kit (900-K60).
- The absorbance of each well was measured at 405 nm and 650 nm after 10 min of incubation with ABTS liquid substrate on a multi-well plate reader .
- The absorbance at 650 nm was subtracted from the absorbance at 405 nm to determine the relative absorbance of each well, and the amount of NGF was calculated from a standard curve.
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-
The amount of NGF released from gels with and without the C6S-binding peptide and C6S was quantified.
- Gels (50 µl) were prepared in triplicate in 2 ml siliconized tubes, as previously described, that contained 2 µg/ml NGF (13257019).
- After the gels were cross-linked, 2 ml of 1xPBS was added onto each gel.
- At each time point, 500 µl of buffer was collected from each tube and immediately replaced with 500 µl of buffer using siliconized pipets and tubes.
- NGF release was monitored over 48 h, and samples were stored at -20°C.
- After 2 d, the gels were broken up with a spatula and digested in 10 units/ml collagenase and 0.4 units/ml chondroitinase ABC for 48 h at 37°C with gentle shaking.
- The amount of NGF in the collected samples was quantified with a human β-NGF ELISA development kit (900-K60).
- The absorbance of each well was measured at 405 nm and 650 nm after 10 min…
-
The amount of NGF released from gels with and without the C6S-binding peptide and C6S was quantified.
- Gels (50 µl) were prepared in triplicate in 2 ml siliconized tubes, as previously described, that contained 2 µg/ml NGF (13257019).
- After the gels were cross-linked, 2 ml of 1xPBS was added onto each gel.
- At each time point, 500 µl of buffer was collected from each tube and immediately replaced with 500 µl of buffer using siliconized pipets and tubes.
- NGF release was monitored over 48 h, and samples were stored at -20°C.
- After 2 d, the gels were broken up with a spatula and digested in 10 units/ml collagenase and 0.4 units/ml chondroitinase ABC for 48 h at 37°C with gentle shaking.
- The amount of NGF in the collected samples was quantified with a human β-NGF ELISA development kit (900-K60).
- The absorbance of each well was measured at 405 nm and 650 nm after 10 min of incubation with ABTS liquid substrate on a multi-well plate reader .
- The absorbance at 650 nm was subtracted from the absorbance at 405 nm to determine the relative absorbance of each well, and the amount of NGF was calculated from a standard curve.
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