RAW264.7 cells were cultured for 5 days on Osteo Assay 24-well plates under differentiation conditions. Acridine orange (3,6-bis[Dimethyloamine] acridine] at 10 μg/ml was loaded for 45 minutes in the culture medium. The dye was rinsed out and the cells were observed by inverted fluorescence microscopy (Leica DMI6000) [31 (link)]. As a control, some cells were treated with the proton pump inhibitor bafilomycin A (200 nM; Enzo Life Science, Farmingdale, NY).
Bafilomycin a
Manufactured by Enzo Life Sciences
Bafilomycin A is a macrolide antibiotic and a specific inhibitor of vacuolar-type H+-ATPase (V-ATPase). It blocks the acidification of organelles by inhibiting the proton pump activity of V-ATPase.
Automatically generated - may contain errors
Lab products found in correlation
Dmi6000, by Leica (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Red blood cell lysis solution, by Miltenyi Biotec (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Resveratrol, by Merck Group (1 mentions)
Tmr dextran, by Thermo Fisher Scientific (1 mentions)
Ultrapure e coli lps, by InvivoGen (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Fatty acids, by Nu-Chek Prep (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
4 protocols using bafilomycin a
1
Osteoblast Differentiation and Acidic Vesicle Assay
2
Thioglycolate-Elicited Peritoneal Macrophage Isolation
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Peritoneal macrophages were harvested by lavage with PBS (Life Technologies), 3 days after injection of 1.5 ml of sterile 4% thioglycolate medium (BD Biosciences) into the peritoneal cavity of the mice. After red blood cells lysis with Red Blood Cell Lysis solution (Miltenyi Biotec), cells were seeded in RPMI supplemented with 10% fetal bovine serum (FBS) and washed after 3 h of adhesion. Conditioned medium experiments were performed by adding centrifuged conditioned medium obtained from AML-12 or Hepa1-6 cells for 6 h. When indicated, cells were treated with 10 μM chloroquine (Sigma), 100 nM bafilomycin A (Enzo Life Sciences), 100 nM rapamycin (Sigma) or 30 μM resveratrol (TCI). When indicated, cells were treated with 10 μM SB202199 in the presence of control medium or conditioned medium from Hepa-1.6 cells for 6 h.
3
Lysosome Dynamics and Macrophage Function
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CAO74-ME and bafilomycin A were from Enzo Life Sciences. Lysotracker red and TMR-dextran (10,000 MW) were from Invitrogen. The cathepsin B activity assay was from Immunocytochemistry Technologies. Ultrapure E. coli LPS was from Invivogen. Thioglycollate was from Difco. Fatty acids were from Nu-Chek Prep. The cathepsin D and LAMP1 antibodies were from Abcam. The actin antibody was from Sigma-Aldrich. The CD107a (LAMP-1) PE conjugated antibody was from eBiosciences (cat#12-1071). The ATG5 antibody was from Novus Biologics. Ultrapure-bovine serum albumin (BSA) was from Lampire and was tested for TLR ligand contamination prior to use.
4
Depletion and Regeneration of Cycling Cells in Planarians
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To deplete cycling cells at 18°C, cohorts of 20 animals were exposed to N-hydroxyurea (HU, Applichem, 5 mM diluted in HM) as indicated in Fig. 2 E. The post-HU antibiotic treatment was given as an antibiotic mix containing ampicillin, rifampicin, streptomycin and neomycin (50 mg/ml each) as reported previously (Wein et al., 2018 (link)). This treatment was given continuously from day 2 after HU release and changed every other day. Untreated animals were kept in HM containing 0.01% DMSO during the same period. For rapamycin treatment, animals were continuously exposed to rapamycin (LC Laboratories, 0.8 µM diluted in HM) from 3 dpt to 10°C, the drug being changed three times a week. To inhibit proteasomal degradation, animals were treated with the proteasome inhibitor MG132 (SelleckChem, 5 µM diluted in HM, DMSO 0.05%) either for 16 h, or over a 5-day period at the indicated concentrations. To test the effect of these drugs on head regeneration, two sets of 15 animals per condition fed twice a week prior to bisection were starved for 2 days, bisected at the mid-gastric position and monitored over 13-15 days. For bafilomycin A (Enzo Life Sciences) treatment, animals were exposed to 50, 100 or 200 nM solution for 16 h diluted in HM, DMSO 0.01%.
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