Female C57BL/6 mice were obtained from Jingda Laboratory Animal Company (Changsha, China). After being anaesthetized with pentobarbital sodium, the mice received an intratracheal injection of 50 μL of bleomycin (BLM) (3.5 mg/kg) (Nippon Kayaku, Japan) on day 0. To study the antifibrotic effects of BM-MSCs pretreated with rmG-CSF (PeproTech, USA), C57BL/6 mice were randomly assigned to one of the following groups: (1) control group, intratracheal saline plus tail vein injection of phosphate-buffered solution (PBS); (2) BLM group, intratracheal BLM plus tail vein injection of PBS; (3) BLM + rmG-CSF- (30 ng/mL) pretreated BM-MSC (1 × 105 in 100 μL) group, intratracheal BLM plus rmG-CSF-pretreated BM-MSC infusion into the tail vein; (4) BLM + rmG-CSF- (30 ng/mL) pretreated BM-MSC (3 × 105 in 100 μL) group; (5) BLM + rmG-CSF- (30 ng/mL) pretreated BM-MSC (1 × 106 in 100 μL) group; (6) BLM + BM-MSC (1 × 105 in 100 μL) group, intratracheal BLM plus BM-MSC infusion into the tail vein; (7) BLM + BM-MSC (3 × 105 in 100 μL) group; and (8) BLM + BM-MSC (1 × 106 in 100 μL) group. rmG-CSF-pretreated BM-MSCs and nontreated BM-MSCs were infused into the tail vein on day 14 after BLM injection, and lung tissues were harvested on day 21.
C57bl 6 mice
Manufactured by Thermo Fisher Scientific
Sourced in United States
C57BL/6 mice are a widely used inbred strain of laboratory mice. They are commonly used as a standard reference strain in biomedical research due to their well-characterized genome and physiological traits.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
B6d2f1 mice, by Jackson ImmunoResearch (3 mentions)
Leibovitz s l 15 medium, by Thermo Fisher Scientific (3 mentions)
C57bl 6, by Jackson ImmunoResearch (3 mentions)
Penicillin g, by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
C57bl 6 mice, by Charles River Laboratories (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Rmg csf, by Thermo Fisher Scientific (1 mentions)
Bleomycin blm, by Nippon Kayaku (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
2 mercaptoenthanol, by Merck Group (1 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Recombinant human m csf, by Thermo Fisher Scientific (1 mentions)
Thioglycollate medium, by Merck Group (1 mentions)
Macrophage colony stimulating factor, by Thermo Fisher Scientific (1 mentions)
Co2 chamber, by Thermo Fisher Scientific (1 mentions)
26 protocols using c57bl 6 mice
1
Antifibrotic Effects of BM-MSCs Pretreated with rmG-CSF in Bleomycin-Induced Lung Fibrosis
2
Murine Bone Marrow-Derived Dendritic Cell Protocol
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Immunocompetent C57bl/6 mice (female, 6–8 weeks old) were purchased from Charles River Laboratories (Wilmington, USA). Animals were housed in a specific pathogen-free (SPF) facility at MIT which is accredited by American Association for Laboratory Animal Care (AALAC). All animal protocols were approved by the MIT Committee on Animal Care (CAC). A human colon cancer cell line, Caco-2, was purchased from ATCC and regularly tested for mycoplasma contamination. Cells were maintained in DMEM media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin). Murine BMDCs were harvested following previously reported protocols (30 (link), 31 (link)). Briefly, bone marrow cells were collected from femurs and tibias from C57bl/6 mice and incubated for 6 days with RPMI media supplemented with 10% FBS, 1% antibiotics (100 μg/ml streptomycin, 100 U/ml penicillin), 40 ng/ml GM-CSF (Peprotech), and 20 ng/ml 2-mercaptoenthanol (Sigma) and harvested on day 7 for in vitro experiments. Cell culture regents were purchased from Thermo Fisher (Waltham, USA), unless otherwise stated.
3
Bone Marrow-Derived Macrophage Isolation
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BMDM obtained from WT or TLR4−/− C57BL/6 mice (The Jackson Laboratory), C3H mice or Flatiron (ffe/+ heterozygous) mice in the C3H genetic background (provided by D. Ward and J. Kaplan, U. Utah) or Hamp−/− C57BL/6 mice (provided by S. Vaulont, Cochin Institite - [27] (link)) were prepared as described [28] (link) and cultured in RPMI 1640 containing 10% heat-inactivated FBS, 100 units/ml penicillin and 100 µg/ml streptomycin and 50 ng/ml human Recombinant M-CSF (Peprotech). Mature, adherent BMDM were detached from dishes with PBS/1 mM EDTA, viability was determined with a trypan-blue exclusion test, cells were seeded into tissue culture-treated 6 well plates at a density of 2×106 cells/well, and incubated overnight at 37°C in a 5% CO2 incubator prior to use in experiments.
4
Isolation and Culture of Diverse Cell Lines
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HEK293T, RAW264.7, and HeLa cells were cultured in DMEM (Hyclone) containing 10% FBS (Gibco) incubated in a 5% CO2 chamber (Thermo Fisher Scientific). BMMs, BMDCs, and mouse pMs were cultured in RPMI 1640 (Gibco) containing 10% FBS. BMMs or BMDCs were derived from bone marrow of 18–20 g C57BL/6 mice (Guangdong Medical Laboratory Animal Center) and cultured for 6–8 d with 100 ng/ml macrophage colony-stimulating factor (PeproTech) or 20 ng/ml granulocyte macrophage colony-stimulating factor (PeproTech). pMs were harvested with 5 ml PBS 3 d after intraperitoneal injection of 1 ml of 4% thioglycollate medium (Sigma-Aldrich). MEFs were derived from embryos staged at embryonic days (E) 13.5–14.5 of gestation of C57BL/6 WT mice. The embryos were cut and enzymatically disaggregated after removing blood and liver tissue to generate single MEF cells, which were then plated at 106 cells per 100-mm dish in DMEM. HEK293T, HEK293T/TLR4, RAW264.7, BMMs, BMDCs, and pMs were treated with 100–200 ng/ml LPS (Sigma-Aldrich). HeLa cells were treated with 1 µg/ml LPS.
5
Antibody-Dependent Cellular Phagocytosis Assay
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Bone marrow cells were obtained from the femurs of C57BL/6 mice (Guangdong Medical Laboratory Animal Center, Guangzhou, China), and induced into mature macrophages using recombinant mouse M-CSF (Peprotech). CHO-K1-PD1 cells were stained with carboxyfluorescein succinimidyl ester (CFSE) (Biolegend) and CFSE+ CHO-K1-PD1 cells were used as target cells. Totally 100 μL each of macrophages (5*102 cells/μL), CFSE+ CHO-K1-PD1 cells (1.5*106 cells/mL), and penpulimab (at the final concentration of 10, 1, and 0.1 μg/mL) or isotype control antibody (at the final concentration of 10 μg/mL) were aliquoted into a 1.5 mL centrifuge tube, respectively. Then, 100 μL APC-labeled goat anti-mouse CD11b antibody (Biolegend) (dilutions 1:400) was added to each sample, and incubated on ice for 40 minutes. After wash with PBS containing 1% bovine serum albumin (BSA) and centrifugation, the cells were resuspended in 200 μL PBS containing 1% BSA and transferred to BD flow sample tube. The ratio of APC+CFSE+ cells to APC+ cells was used as the phagocytic rate to evaluate antibody-mediated ADCP activity. ADCP% was calculated as follows: ADCP% = ((number of APC+CFSE+ cells)/(number of APC+ cells)) * 100%.
6
Isolation of Murine Bone Marrow Cells
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C57BL/6 mice were purchased from Charles River UK and housed in the Beatson Biological Service and Research Units. All mouse experiments were approved by the local animal welfare ethical review body (AWERB) committee and United Kingdom (UK) home office and performed according to UK Home Office project license 60/4512 approved 25 April 2013(Animal Scientific Procedures Act 1986) guidelines. Femur, tibia and pelvic girdle of wild-type (WT) C57BL/6 mice were crushed in phosphate buffered saline (PBS)/2% FBS and passed through a 40-μM cell strainer (Fisher Scientific, Loughborough, UK) to harvest BM. C-kit enrichment was carried out by magnetic activated cell sorting (MACS) separation of BM samples following incubation with anti-CD117 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany).
7
Evaluating TLR4 Activation and Dendritic Cell Response
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To determine TLR4 activation, HEK-Blue™ TLR4 cells (Invivogen) were incubated with increasing concentrations of MPLA PS for 24 h 37 °C in a 5% CO2 incubator. NF-kB-induced SEAP activity was detected using QUANTI-Blue™ (Invivogen) and by reading the OD at 650 nm. For dendritic cell activation experiments, BMDCs were prepared from C57Bl/6 mice (Jackson) as previously described.66 (link) After 9 days of culture, cells were seeded at 2 × 105 cells/well in round-bottom 96-well plates (Fisher Scientific) in IMDM with 10% FBS and 2% penicillin/streptomycin (Life Technologies). Cells were treated with varying concentrations of MPLA PS or free MPLA and incubated for 24 h at 37 °C in a 5% CO2 incubator. After 24 h, the supernatant was collected, and cytokine concentration was measured using a multiplexed mouse Th cytokine panel (BioLegend) according to the manufacturer’s instructions.
8
Bone Marrow Transplantation in Mice
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Example 76
Female C57BL/6 and B6D2F1 mice are purchased from the Jackson Laboratory (Bar Harbor, Me., USA). Bone marrow is harvested from the femurs and tibias of 12-20 week old mice. Before receiving transplantation, B6D2F1 mice are given 14 Gy total body irradiation (137Cs source). Irradiation is done twice, three hours apart. 5×10{circumflex over ( )}6 bone marrow cells are supplemented with 2×10{circumflex over ( )}6 nylon-wool nonadherent splenetic T cells from C57BL/6 mice and resuspended in Leibovitz's L15 medium (Life Technologies Inc., New York USA) and transplanted by tail vein infusion (0.25 mL) into B6D2F1 mice (Cooke K R, Gerbitz A, Crawford J M, Teshmia T, Hill G R, Tesolin A. Rossignol D P, Ferrara J L M, 2001. LPS antagonism reduces graft-versus-host disease and preserves graft-versus-host leukemia after experimental bone marrow transplantation. The Journal of Clinical Investigation. 107:1581-1589).
9
Bone Marrow Transplant in Mice
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Example 76
Female C57BL/6 and B6D2F1 mice are purchased from the Jackson Laboratory (Bar Harbor, Me., USA). Bone marrow is harvested from the femurs and tibias of 12-20 week old mice. Before receiving transplantation, B6D2F1 mice are given 14 Gy total body irradiation (137Cs source). Irradiation is done twice, three hours apart. 5×10 6 bone marrow cells are supplemented with 2×10 6 nylon-wool nonadherent splenetic T cells from C57BL/6 mice and resuspended in Leibovitz's L15 medium (Life Technologies Inc., New York USA) and transplanted by tail vein infusion (0.25 mL) into B6D2F1 mice (Cooke K R, Gerbitz A, Crawford J M, Teshmia T, Hill G R, Tesolin A, Rossignol D P, Ferrara J L M 2001. LPS antagonism reduces graft-versus-host disease and preserves graft-versus-host leukemia after experimental bone marrow transplantation. The Journal of Clinical Investigation. 107:1581-1589).
10
Bone Marrow Transplant in Mice
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Example 76
Female C57BL/6 and B6D2F1 mice are purchased from the Jackson Laboratory (Bar Harbor, Me., USA). Bone marrow is harvested from the femurs and tibias of 12-20 week old mice. Before receiving transplantation, B6D2F1 mice are given 14 Gy total body irradiation 137Cs source). Irradiation is done twice, three hours apart. 5×10{circumflex over ( )}6 bone marrow cells are supplemented with 2×10{circumflex over ( )}6 nylon-wool nonadherent splenetic T cells from C57BL/6 mice and resuspended in Leibovitz's L15 medium (Life Technologies Inc., New York USA) and transplanted by tail vein infusion (0.25 mL) into B6D2F1 mice (Cooke K R, Gerbitz A, Crawford J M, Teshmia T, Hill G R, fesolin A, Rossignol D P, Ferrara J L M, 2001. LPS antagonism reduces graft-versus-host disease and preserves graft-versus-host leukemia after experimental bone marrow transplantation. The Journal of Clinical Investigation. 107:1581-1589).
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