Part:BBa_K3562012
CXCL3
Growth-regulated gamma protein(GRO-gamma) that has chemotactic activity for neutrophils.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Chemokines are a family of small chemotactic cytokines, and the name is derived from their ability to induce directed chemotaxis in nearby responsive cells. All chemokines possess a number of conserved cysteine residues involved in intramolecular disulfide bond formation. Some chemokines can be induced during an immune response to recruit cells of the immune system to a site of infection, while others are considered homeostatic and are involved in controlling the migration of cells during normal processes of tissue maintenance or development. Chemokines are found in all vertebrates, some viruses and some bacteria.
GRO-gamma/CXCL3Ligand for CXCR2 (By similarity). Has chemotactic activity for neutrophils. May play a role in inflammation and exert its effects on endothelial cells in an autocrine fashion. In vitro, the processed form GRO-gamma(5-73) shows a fivefold higher chemotactic activity for neutrophilic granulocytes.
Protocols
A. Preparation of chemokine mother liquor
1. Short centrifuge chemokine dry powder
2. The mother liquor of 7 chemokines was prepared:
6.33 μl (CXCL1, cxcl2, cxcl3)
5.95 μl（CXCL8）
5.75 μl（CCL2）
6.41 μl（CCL3）
5.62 μl（CCL8）
The mother liquor of 1E5 nmol / L of seven chemokines was obtained
3. 1 μl mother liquor of each chemokine was added into the EP tube containing 999 μl medium to obtain 1 μl dilution
4. Dilution 1 of each chemokine: 200 μl was added into the EP tube containing 1800 μl medium to obtain dilution 2 of 10 nmol / l
B. Cell suspension was prepared
1. Select 5 THP1 culture dishes, take out the total volume of 25 ml cell suspension, take out 10 μl suspension for cell counting plate count, estimate cell density and suspension volume
2. 800 rpm, 5 min centrifugation, discard the supernatant, add 8.4 ml medium to resuspend cells
C. Transwell Migration Assay
1. 200 μl cell suspension was added into the upper chamber of each small hole, and 600 μl medium or chemokine solution was added into the lower chamber
2. Put it into the incubator for overnight cultivation
D. Flow cytometry protocol
1. At 17:47, Transwell plate was taken out to prepare for flow cytometry (cytflex) The treated cell culture medium was collected to the special flow tube;
2. Add 1ml PBS buffer for cleaning once, and add the cleaning solution into the tube;
3. The cells were collected by trypsin and centrifuged at 1000xg for 5 min to remove the supernatant;
4. 1 ml PBS buffer was used to wash the remaining cells once, and the cleaning solution was transferred to the centrifuge tube;
Note: 3-6 steps are collected from the same first-class special pipe.</p>
5. Centrifugation at 1000xg for 5 min, and discard the supernatant;
6. Cells were resuspended with 1 ml PBS buffer and centrifuged again to discard the supernatant;
7. One hour internal flow cytometry was used to detect the number of cells, and nylon mesh was used to filter before operation.
Results
We used the Transwell experiment combined with flow cytometry to verify the chemotactic ability of this chemokine on the THP1 cell line.(Fig.1)
Besides, we also tested the activity of six other chemokines.(Fig.2)
Reference
[1]Chemokine activity definition
[2]UniProtKB - P19876 (CXCL3_HUMAN)
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