Mice were injected with 4% sterile thioglycolate intraperitoneally. After 4 h, mice were injected with 4% sterile thioglycolate (Sigma-Aldrich, B2551) intraperitoneally. After 4 h, mice were euthanized, and the peritoneal space was flushed twice with 10 mL of sterile PBS to harvest neutrophils. Cells were washed in PBS and counted with a hematocytometer.
B2551
Manufactured by Merck Group
The B2551 is a laboratory centrifuge device designed for separating and isolating different components of a liquid sample. It is capable of generating high centrifugal forces to facilitate the separation process. The core function of the B2551 is to enable efficient and accurate separation of substances based on their densities.
Automatically generated - may contain errors
Lab products found in correlation
Lps b5 ultrapure, by InvivoGen (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Polyethyleneimine, by Thermo Fisher Scientific (1 mentions)
D5796, by Merck Group (1 mentions)
4 octyl itaconate, by Merck Group (1 mentions)
Ki696, by MedChemExpress (1 mentions)
Middlebrook 7h9 medium, by BD (1 mentions)
3 protocols using b2551
1
Neutrophil Isolation from Thioglycolate-Induced Mice
2
Macrophage Polarization Modulation
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L929 cells were obtained from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium (DMEM; D5796, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; 97068-085, VWR). BMDMs were generated from bone marrow and cultured on petri plates in DMEM containing 10% FBS and 30% L929 culture media for 7 days. PerMacs were collected from peritoneal gavages 4 days after intraperitoneal injection of 3% brewer thioglycolate medium (B2551, Sigma-Aldrich). Transfection of ISD (InvivoGen) into the cytosol of BMDMs was performed using polyethyleneimine (43896, Alfa Aesar). Unless stated, 6 × 105 BMDMs and 1.2 × 106 PerMacs per milliliter were used in in vitro experiments. The LPS-B5 Ultrapure (InvivoGen) concentration used was 200 ng/ml for BMDMs and 20 ng/ml for PerMacs unless otherwise indicated. The 4-octyl-itaconate (Cayman) concentration used was 125 μM, the DMF (Sigma-Aldrich) concentration was 50 μM, and the KI696 (MedChemExpress) concentration was 20 μM. Cells were exposed to both treatments 6 hours before LPS stimulation.
3
Isolation and Infection of Peritoneal Macrophages
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Peritoneal macrophages were isolated as described61 (link). Briefly, mice were injected intraperitoneally (IP) with 2.0 mL of 4% Brewer’s thioglycollate medium (B2551, Sigma-Aldrich). After 3 days, primary macrophages in the peritoneal lavage were collected from euthanized animals using 10 mL cold PBS. Subsequently, the cells were plated in 12-well plates at 106 cells/well in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin and were incubated at 37 °C in 5% CO2 for 2 h. The cultures were then washed three times with PBS to remove non-adherent cells, and the remaining adherent monolayer cells were used as primary peritoneal macrophages.
Mtb H37Rv (H37Rv) were grown to mid-log phase in Middle brook 7H9 medium (271310, Becton Dickinson, Cockeysville, MD) with 0.05% Tween-80 and 10% oleic acid-albumin-dextrose-catalase (OADC) enrichment (211886, Becton Dickinson, Sparks, MD). Before infection, Mtb were suspended in complete medium without antibiotics. Peritoneal macrophages were infected with H37Rv at a MOI of 5.
Mtb H37Rv (H37Rv) were grown to mid-log phase in Middle brook 7H9 medium (271310, Becton Dickinson, Cockeysville, MD) with 0.05% Tween-80 and 10% oleic acid-albumin-dextrose-catalase (OADC) enrichment (211886, Becton Dickinson, Sparks, MD). Before infection, Mtb were suspended in complete medium without antibiotics. Peritoneal macrophages were infected with H37Rv at a MOI of 5.
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