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Recombinant mouse ccl2

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Recombinant mouse CCL2 is a recombinant protein that represents the mouse chemokine (C-C motif) ligand 2. It is a secreted chemokine that plays a role in the recruitment and activation of monocytes and macrophages.

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Lab products found in correlation

Dnase 1, by Merck Group (2 mentions) Cytoselect cell migration assay kit, by Cell Biolabs (1 mentions) 96 well transwell plate, by Corning (1 mentions) Celltracker green, by Thermo Fisher Scientific (1 mentions) Recombinant mouse ccl5, by BioLegend (1 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)

5 protocols using recombinant mouse ccl2

1

Colonic Lamina Propria Isolation and Blood Leukocyte Stimulation

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Isolation of colonic lamina propria cells was adapted from a previously described protocol (68 ). After dissociation of the epithelial layer for 10 minutes x3 at room temp, remaining colon tissue was minced using scissors and digested in media with 500ug/mL of DNASE I (Sigma Aldrich) and 1mg/mL of Collagenase type IV (Worthington Biochemical Company). Digested tissue was filtered sequentially through 100 μm and 40 μm filters followed by centrifugation and resuspension in buffer for subsequent staining. Blood leukocytes were isolated after retro-orbital collection of whole blood, followed by RBC lysis and staining for flow cytometry. In one experiment, fresh blood leukocytes were exposed to recombinant mouse CCL2 (Biolegend) in RPMI complete media (RPMI-1640, 10%FBS, 1%Pen/Strep) and incubated in 95% air with 5% CO2 at 37°C for 1 hour.
Schaefer R.E., Callahan R.C., Atif S.M., Orlicky D.J., Cartwright I.M., Fontenot A.P., Colgan S.P, & Onyiah J.C. (2021). Disruption of monocyte-macrophage differentiation and trafficking by a heme analog during active inflammation. Mucosal immunology, 15(2), 244-256.
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2

Cell Migration Assay for T cells and Macrophages

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Cell migration a was determined using the CytoSelect Cell Migration Assay kit (Cell Biolabs, San Diego, CA, USA), according to the manufacture’s protocol. Briefly, T cells (CD4+ cells, 1 × 106) from the spleen and macrophages (F4/80+ cells, 1 × 106) from the liver were isolated, and cells were loaded into the upper chamber of the two systems. The lower chambers were loaded with 300 ng/ml recombinant mouse CCL2 (BioLegend) or vehicle (PBS). After 6 h of stimulation, cells that had migrated were stained using CyQuant GR dye solution, and fluorescence intensity was measured at 480 nm/520 nm. Data are shown in relative fluorescence units.
Inoue T., Ito Y., Nishizawa N., Eshima K., Kojo K., Otaka F., Betto T., Yamane S., Tsujikawa K., Koizumi W, & Majima M. (2018). RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis. PLoS ONE, 13(11), e0200432.
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3

Colonic Lamina Propria Isolation and Blood Leukocyte Stimulation

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Isolation of colonic lamina propria cells was adapted from a previously described protocol (68 ). After dissociation of the epithelial layer for 10 minutes x3 at room temp, remaining colon tissue was minced using scissors and digested in media with 500ug/mL of DNASE I (Sigma Aldrich) and 1mg/mL of Collagenase type IV (Worthington Biochemical Company). Digested tissue was filtered sequentially through 100 μm and 40 μm filters followed by centrifugation and resuspension in buffer for subsequent staining. Blood leukocytes were isolated after retro-orbital collection of whole blood, followed by RBC lysis and staining for flow cytometry. In one experiment, fresh blood leukocytes were exposed to recombinant mouse CCL2 (Biolegend) in RPMI complete media (RPMI-1640, 10%FBS, 1%Pen/Strep) and incubated in 95% air with 5% CO2 at 37°C for 1 hour.
Schaefer R.E., Callahan R.C., Atif S.M., Orlicky D.J., Cartwright I.M., Fontenot A.P., Colgan S.P, & Onyiah J.C. (2021). Disruption of monocyte-macrophage differentiation and trafficking by a heme analog during active inflammation. Mucosal immunology, 15(2), 244-256.
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4

Transwell Assay for Myeloid Cell Migration

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BPR cells were cultured in DMEM-F12 supplemented with FBS, then switched to serum-free media. Forty-eight hours later, BPR-conditioned medium (CM) was harvested. DMEM-F12 or CM (150 µl), with or without CCL2 blocking antibody (3µg/mL, Clone 123616, R&D Systems), was added to the bottom chamber of a 96-well transwell plate (Corning), and incubated overnight at 4°C. RBC-depleted splenocytes (5×105) resuspended in 50µL DMEM-F12 were placed in the top chamber of the transwell insert, and allowed to migrate for 5 hours at 37°C before cells from top and bottom chambers were harvested, stained for CD45, CD11b, Ly6C, and Ly6G, and populations were quantified by flow cytometry. To assess migration towards CCL2, 20ng/ml recombinant mouse CCL2 (Biolegend) was added to 0.5% FBS-enriched DMEM:F12 before incubating in the bottom transwell chamber overnight. RBC-depleted splenocytes resuspended in 0.5% FBS-enriched DMEM:F12 media were placed in the top chamber and allowed to migrate for 18 hours before being harvested and analyzed as described. Migration index was calculated as the number of cells with the indicated phenotype in the bottom chamber, divided by the total number of such cells in both chambers.
Steinberg S.M., Shabaneh T.B., Zhang P., Martyanov V., Li Z., Malik B.T., Wood T.A., Boni A., Molodtsov A., Angeles C.V., Curiel T.J., Whitfield M.L, & Turk M.J. (2017). Myeloid cells that impair immunotherapy are restored in melanomas which acquire resistance to BRAF inhibitors. Cancer research, 77(7), 1599-1610.
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5

Chemotaxis of Polarized Th1 Cells

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In vitro polarized Th1 cells were labeled with 1 μM CellTracker Green (CMFDA Dye, Life Technologies) at 37°C with 5% CO2 for 30 min. Chemotactic behavior was assessed as previously described (Nishihara et al., 2020 (link)) by allowing 105 Th1 cells to migrate for 2 h across laminin (from Engelbreth–Holm–Swarm murine sarcoma basement membrane, Sigma) coated Millicell filters (pore size 5.0 μm, pore density 2.0×106 pores/cm2, growth area 0.33 cm2; Millicell, MCMP24H48) with 0, 1, 10, 100 or 1000 ng/ml recombinant mouse CCL2 (Biolegend) or recombinant mouse CCL5 (Biolegend) in migration assay medium (DMEM, 5% FBS, 4 mM L-glutamine and 25 mM HEPES) in the bottom compartment. Migrated Th1 cells were collected and counted with an Attune NxT Flow Cytometer (Thermo Fisher Scientific) by gating on CMFDA-positive live cells.
Castro Dias M., Odriozola Quesada A., Soldati S., Bösch F., Gruber I., Hildbrand T., Sönmez D., Khire T., Witz G., McGrath J.L., Piontek J., Kondoh M., Deutsch U., Zuber B, & Engelhardt B. (2021). Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood–brain barrier. Journal of Cell Science, 134(8), jcs253880.
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