The inflammasome reporter macrophages stably transduced with constructs for the expression of mCerulean or mCherry-tagged ASC have been described14 (link). Bone-marrow derived macrophages (BMDMs) were obtained by culturing bone marrow cells from 6- to 8-week old C57BL/6 mice in DMEM supplemented with 10% FBS, 10 μg/ml Ciprobay-500 and 40 ng/ml M-CSF (R&D Systems). Six days later, BMDMs were collected and plated. Immortalized BMDMs were cultured in DMEM supplemented with 10% FBS and 10 μg/ml Ciprobay-500. Human peripheral blood mononuclear cells (PBMCs) were purified from whole blood over Ficoll density gradients (GE Healthcare); erythrocytes were lysed in red cell lysis buffer (Miltenyi Biotec) and seeded in RPMI supplemented with 10% FBS and 10 μg/ml Ciprobay-500. The monocytic cell line THP1 was cultured in RPMI 1640 supplemented with 10% FBS and 10 μg/ml Ciprobay-500. For stimulation assays, cells were treated with 100 nM of phorbol 12-myristate 13-acetate (PMA) overnight, primed with 1 μg/ml of LPS for 2 h and further activated with 10 μM of nigericin for the time indicated in figure legends.
Red cell lysis buffer
Manufactured by Miltenyi Biotec
Red cell lysis buffer is a solution designed for the selective lysis of erythrocytes (red blood cells) in biological samples, such as blood or bone marrow. Its core function is to facilitate the removal of red blood cells from the sample, enabling the isolation and analysis of other cell types of interest, such as leukocytes (white blood cells).
Automatically generated - may contain errors
Lab products found in correlation
Ciprobay 500, by R&D Systems (1 mentions)
Ficoll density gradient, by GE Healthcare (1 mentions)
M csf, by R&D Systems (1 mentions)
Macs dead cell removal kit, by Miltenyi Biotec (1 mentions)
40 μm nylon mesh, by Thermo Fisher Scientific (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
3 protocols using red cell lysis buffer
1
Isolation and Activation of Macrophages
2
Isolation of Murine Uterine Epithelial Cells
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Mouse uteri were surgically removed and minced using scissors. Tissues were digested using the MACS Multi Tissue Dissociation Kit II (Miltenyi Biotec) for 80 min at 37 °C. Digested tissues were strained through a 40 μm nylon mesh (ThermoFisher). The Red Cell Lysis Buffer (Miltenyi Biotec) was used to remove red blood cells. Dead cells removed using the MACS Dead Cell Removal Kit (Miltenyi Biotec), and EPCAM-positive cells were positively selected and purified using a PE-conjugated EPCAM antibody and anti-PE MicroBeads (Miltenyi Biotec), per the manufacturers’ instructions. A BD Accuri C6 flow cytometer (BD Biosciences) was used to confirm purity of EPCAM-positive population.
3
Purification of Primary Hepatocytes
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Hepatocytes were purified from fresh liver. The liver was harvested into ice-cold RPMI media and subsequently placed into liver digestion media (Hanks’ Balanced Salt Solution (HBSS) without calcium or magnesium, supplemented with 120 U.mL−1 Type III collagenase) and incubated at 37 °C for 30 min with gentle agitation. The liver was then homogenised, passed through a 70-μm cell strainer, and the digestion media inactivated with the addition of 5 mL blocking buffer (HBSS supplemented with 5 mM EDTA and 2% v/v FBS). Samples were centrifuged and resuspended in 5 mL ice-cold red cell lysis buffer (MACS Miltenyi Biotec) and incubated for 3 min on ice. Samples were diluted with 25 mL RPMI/5% v/v FCS and passed through a 70-μm cell strainer, and centrifuged. Cell pellets were resuspended in 5 mL RPMI/5% v/v FCS and layered over a 10 mL LymphoprepTM cushion, centrifuged at 800 × g for 25 min at 20 °C. The cell pellet containing mainly hepatocytes was transferred to a fresh 15 mL Falcon tube and washed three times in 5 mL RPMI/5% v/v FCS for 5 min. Finally, hepatocytes were resuspended in RPMI/5% v/v FCS and mixed at a 1:1 ratio with isotonic Percoll® (1.07 g.mL−1) and centrifuged at 800 × g for 5 min. Subsequently, the upper fraction containing mainly hepatocytes was collected, and washed in PBS, and cell pellets were stored at −80 °C until further analysis.
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