Bone marrow was obtained from femurs and tibia of female wild-type mice and cultured for 6–8 d in RPMI containing 8% FCS, 100 IU/mL penicillin, 100 μg/mL streptomycin (Invitrogen) supplemented with 20 ng/ml murine CSF-1 (Peprotech, 315-02). For experiments, BMDM were primed overnight in medium containing 100 ng/ml LPS. The next day, BMDM were washed and cultured in control RPMI or RPMI 1:1 supplemented with conditioned medium from KEP tumor cell lines (KEPCM) with or without anti-CCL2 (5 μg/ml). KEP tumor cell line-conditioned media was generated by culturing KEP tumor cell lines in serum-free RPMI for 48 hours. Supernatant was centrifuged to exclude cellular debris before use in BMDM experiments. After 24 hours, BMDM were collected and RNA was extracted using RNeasy columns (Qiagen, 74104). Il1β expression levels were determined by RT-PCR. Fold change was calculated using the formula 2−(ΔCt — X[ΔCtWT]).
Murine csf 1
Manufactured by Thermo Fisher Scientific
Murine CSF-1 is a recombinant protein that functions as a growth factor for the differentiation and proliferation of macrophages in mice. It is often used in cell biology research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Rneasy column, by Qiagen (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Murine gm csf, by Thermo Fisher Scientific (1 mentions)
Lm001 05, by Welgene (1 mentions)
Rbc lysis buffer, by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Murine il 2, by Thermo Fisher Scientific (1 mentions)
4 protocols using murine csf 1
1
Bone Marrow-Derived Macrophage Activation
2
Murine Bone Marrow-Derived Dendritic Cells and Macrophages
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Six-week-old female C57BL/6 mice were used as a source of bone marrow (BM) for the generation of mouse DCs and macrophages in culture as we described previously [51 (link)]. Briefly, BM cells were isolated by flushing femurs and tibiae with PBS. Pelleted cells were resuspended briefly in water to lyse red blood cells and stabilized by adding complete medium (RPMI 1640, 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine). The cells were centrifuged and resuspended in complete medium supplemented with murine CSF1 (100 ng/ml; Peprotech, Rocky Hill, NJ) to grow macrophages, while to grow DCs the media was supplemented with murine GM-CSF (100 ng/ml; Peprotech). The cells were plated in non-tissue culture plastic petri dishes (1 bone per 10 cm dish) for 5 d at 37°C with CO2. After 5 d, the media was removed, and the adherent cells recovered by incubating the cells for 5 min at 37°C with Versene (Invitrogen, San Diego, CA). Cells were washed, counted, and plated onto tissue culture dishes for use the following day.
3
Culturing Murine Bone Marrow Macrophages
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Bone marrow cells were flushed from the femurs and tibias of mice and then depleted of RBCs using RBC lysis buffer (Sigma-Aldrich, R7757). The cells were then cultured in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, LM001-05) supplemented with 10% FBS and 20 ng/ml murine CSF1 (Peprotech, 315–02). Non-adherent cells were carefully removed, and fresh medium was added every 2 d. On day 6, the cells were collected for experiments [52 (link)].
4
Macrophage-mediated CD8+ T cell suppression
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A macrophage-mediated suppression assay on CD8+ T cell proliferation was performed as described previously with modifications (27). CD8+ T cells were isolated from the spleens of C57BL/6/J mice using a negative selection Mojosort kit (BioLegend) and labeled with 5 mg/mL CFSE (Life Technology) for 10 min at 37°C protected from light. Cells were washed twice and resuspended in medium containing 10 ng/mL murine IL-2 (Peprotech) and 10 ng/mL murine CSF-1 (Peprotech). Then, cells were seeded on wells coated with anti-CD3 and anti-CD28 antibodies (2 mg/mL) to allow activation alone or at a ratio of 2:1 (2-CD8T:1-BMDM) with BMDMs previously treated for 24 h with 10 ng/mL IL-4 (Peprotech). During coculture, either CSF-1R Ab (CD115, Invitrogen), rIL-10-Fc or BF10 was added at a concentration of 50 ng/mL. After 72 h, CD8 T cells were collected, stained and analyzed by flow cytometry.
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