Migration assay for myeloid monocytes, RAW264.7, ANA-1, and LLC cells was performed in a transwell Boyden chamber (BD Biosciences). Cell suspension (3×105 cells/mL) was placed in the upper chamber. The lower compartment contained 0.6 mL of recombinant murine CCL2 (Peprotech) or indicated CM. After 24 hours of incubation at 37°C, myeloid monocytes in the lower chamber were counted with Fuchs-Rosenthal counting chamber. RAW264.7, ANA-1, and LLC cells under the upper chamber were fixed with methanol and stained with 0.5% crystal violet (Beyotime) for 20 minutes. The stained cells were subsequently photographed and counted using a microscope (Olympus UIS2; ×200 magnification, at least three random fields/well).14 (link)
Recombinant murine ccl2
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Recombinant murine CCL2 is a chemokine that functions in the recruitment and activation of monocytes and macrophages. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant murine cxcl1, by Thermo Fisher Scientific (2 mentions)
Transwell boyden chamber, by BD (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Ccl2 antibody, by Santa Cruz Biotechnology (1 mentions)
Transwell chamber, by Corning (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Recombinant murine il 23, by Thermo Fisher Scientific (1 mentions)
Recombinant murine il 1β, by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Tisseel, by Baxter (1 mentions)
9 protocols using recombinant murine ccl2
1
Myeloid Monocyte Migration Assay
2
VSMC Migration Assay with Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transwell assay was performed to evaluate VSMC migration in DMEM supplemented with 10% fetal bovine serum (FBS) in the presence or absence of macrophages in a transwell chamber (8-μm pore size; Corning). Briefly, macrophages were seeded into the lower chamber in DMEM medium containing 10% FBS and TAK-242, or CCL2 antibody (100 ng/ml, Santa Cruz, Santa Cruz, CA) that neutralized macrophage-induced migration. VSMCs (104/well) were placed into the upper chamber in DMEM medium or DMEM containing 0.2% bovine serum albumin (BSA).
Recombinant Murine CCL2 (1–100 ng/ml, PeproTech 250–10, Rocky Hill, NJ) was added into the lower chamber. Cells on the filter were removed after 10 hours or 24 hours, and the cells in the bottom chamber were fixed and stained with DAPI. The number of cells that migrated across the filter was counted in five random fields (magnification × 100) using a fluorescence microscope.
Recombinant Murine CCL2 (1–100 ng/ml, PeproTech 250–10, Rocky Hill, NJ) was added into the lower chamber. Cells on the filter were removed after 10 hours or 24 hours, and the cells in the bottom chamber were fixed and stained with DAPI. The number of cells that migrated across the filter was counted in five random fields (magnification × 100) using a fluorescence microscope.
3
Chemotaxis Assay for Macrophage Migration
4
Activation and Cytokine Profiling of γδ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sorted γδ T cells were cultured 1:1 with irradiated splenocytes (40 Gy) in flat bottom 96-wells tissue culture plate (Thermo Scientific) in IMDM containing 8% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin (Invitrogen) and 0.5% β-mercaptoethanol. T cells were activated by addition of Dynabeads Mouse T-activation CD3/CD28 beads (Thermo Scientific, 11456D). Culture medium was supplemented with recombinant murine IL-23 (10 ng/mL; purified by the NKI protein facility) or 50 ng/mL recombinant murine CCL2 (Peprotech, 250-10). After 48 hours of culture, supernatant was collected and stored in -20°C until further use.
5
Chemotaxis Assay to Evaluate Macrophage Migration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell chemotaxis assays were carried out using 8.0 μm pore size transwells (Corning, Corning, NY, USA). CT26 cells were exposed to 12 Gy irradiation, and then the cells were cultured for 24 hours with 40 μmol/L rosiglitazone. Approximately 5 × 104 RAW264.7 cells were resuspended in serum‐free media and seeded in the upper chambers of 24‐well transwells. Serum‐free medium (no chemoattractant) in the lower chambers served as background control. To the remaining lower wells, 10 ng/mL recombinant murine CCL2 (PeproTech Inc., Rocky Hill, NJ, USA) was added. The relevant interventions were used as corresponding conditioned media. After 6 hours of incubation at 37°C, the migrated cells attached to the lower surface of the filters were stained with Wright's Stain Kit. Migrated cells were examined and counted in 5 random microscopic fields. Chemotaxis index was defined as the mean number of migrated RAW264.7 cells in response to conditioned medium as fold increase relative to serum‐free medium control.
6
Modulating Cytokine Levels in Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For CCL2 rescue experiments, female wild-type or Tcrd—/— mice (10-12 weeks of age) were injected intravenously (i.v.) with 1 μg/day recombinant murine CCL2 (Peprotech, 250-10) in 100 μl sterile PBS or vehicle for 5 consecutive days. On the last day animals were sacrificed 1 hr after rCCL2 or vehicle administration and blood and lungs were collected and processed for flow cytometric analysis. For the IL17+ γδ T cell read out, lung and blood cells were pooled to gain sufficient amounts of cells. For neutrophil and monocytes read-out, only blood was used.
For IL1β rescue experiments, mammary tumor-bearing KEP animals were treated twice weekly with anti-CCL2 (C1142 Janssen Pharmaceuticals) by intraperitoneal injection dosed at 10 mg/kg starting from a tumor size of 25 mm2 until animals were sacrificed. When tumors reached a size of ∼130 mm2 animals were injected intraperitoneally (i.p.) with 0,5 μg/day recombinant murine IL1β (Peprotech, 211-11B) for 3 consecutive days. Animals were sacrificed 24 hrs after the last injection and organs were collected and processed for flow cytometry.
For IL1β rescue experiments, mammary tumor-bearing KEP animals were treated twice weekly with anti-CCL2 (C1142 Janssen Pharmaceuticals) by intraperitoneal injection dosed at 10 mg/kg starting from a tumor size of 25 mm2 until animals were sacrificed. When tumors reached a size of ∼130 mm2 animals were injected intraperitoneally (i.p.) with 0,5 μg/day recombinant murine IL1β (Peprotech, 211-11B) for 3 consecutive days. Animals were sacrificed 24 hrs after the last injection and organs were collected and processed for flow cytometry.
7
Preclinical Immunotherapy Combination Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-mouse PD-1 (clone RMP1-14), anti-mouse CTLA-4 (clone 9D9), anti-mouse CD80 (clone 16-10A1), anti-mouse CD86 (clone GL-1), anti-mouse Ly6C (clone Monts 1), anti-mouse CCL2 (clone 2H5), and the isotype-matched IgG controls were purchased from BioXCell. Recombinant murine CCL2 (catalog #250-10), IFN-γ (catalog #315-05), IL4 (catalog #214-14), GM-CSF (catalog #315-03), M-CSF (catalog #315-02) were purchased from PeproTech. Murine lung cancer cell lines 393P, 412P, 344P, 344SQ, and 307P were derived from K-rasLA1/+p53R172HΔg/+ mice as previously described in multiple prior lung cancer studies (16 (link)–23 (link)). The LLC-JSP Lewis lung tumor cell line as well as the B16 and B16-OVA melanoma cell lines were maintained in our laboratories (24 (link), 25 ).
8
Chemokine-Loaded Fibrin Sealant for Wound Healing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fibrin sealant (Tisseel, Baxter) was delivered to the wound bed by co-application of thrombin and fibrinogen, which were prepared under sterile conditions according to the manufacturer instructions. The sealer protein protease inhibitor was omitted from the mixture to facilitate delivery of the recombinant chemokines to the wound bed via fibrin degradation. Directly before application, recombinant murine CCL2 (Peprotech) and recombinant murine CXCL1 (Peprotech) were mixed into the fibrinogen component. Control mice were treated with Tisseel without chemokines. The fibrinogen and thrombin components were maintained at 37°C to avoid polymerization. Using two pipets, equal volumes of fibrinogen and thrombin were simultaneously applied to the wound beds of anesthetized mice and allowed to polymerize. Treatments were given every day from wound days 1 to 7, then every other day for the remainder of the experiment. Each chemokine treatment contained 10ng of recombinant CCL2 and 10ng of recombinant CXCL1.
Application volumes were adjusted according to wound bed area and ranged from 30uL to 10uL.
Application volumes were adjusted according to wound bed area and ranged from 30uL to 10uL.
9
Chemokine-Induced Sponge Angiogenesis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant murine CCL2 (Peprotech) and recombinant murine CXCL1 (Peprotech) were diluted in 1x PBS for injection into implanted PVA sponges. The backs of mice were cleaned with iodine solution and isopropyl alcohol. 0.5ug of each chemokine mixed in a total volume of 50uL of PBS was injected through the skin and into the center of each sponge for a total treatment of 3ug per wound. Control mice received injections of PBS vehicle. Mice were injected on wound days 5 and 6, and sponges were isolated on wound day 7.
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!