NIH 3T3 cells (1 × 107 cells/ml) were treated with 300 U/ml streptolysin O (SLO, Sigma-Aldrich, Budapest) in the presence of 10 mM DL-Dithiothreitol (DTT), protease inhibitor cocktail (Sigma-Aldrich, Budapest, Hungary) and 100 μg/ml phenylmethanesulfonyl fluoride (PMSF) in phosphate-buffered saline (PBS) containing 1% fetal bovine serum (FBS-PBS) at 37°C for 30 min, allowing permeabilization of approximately 60–80% of the cells (judged by propidium iodide (PI) staining). The reaction was stopped with 10 ml ice-cold FBS-PBS and the cells were centrifuged for 5 min at 525 × g at 4°C. Unbound toxin was removed by washing the cells 3 times with FBS-PBS and the cell pellet was re-suspended in FBS-PBS.
Streptolysin o slo
Manufactured by Merck Group
Sourced in Hungary, Germany
Streptolysin O (SLO) is a bacterial toxin produced by certain strains of Streptococcus bacteria. It functions as a pore-forming protein, capable of creating openings in the cell membranes of target cells. SLO is commonly used in research applications as a tool for studying cell membrane dynamics and permeability.
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Lab products found in correlation
Pbs ca2 mg2, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Tnfα duoset elisa kit, by R&D Systems (2 mentions)
Il 1β duoset elisa kit, by R&D Systems (2 mentions)
Z wehd fmk, by R&D Systems (2 mentions)
Caspase 1 mouse matched pair detection set, by Adipogen (2 mentions)
Cathepsin b inhibitor ca 074 me, by Enzo Life Sciences (2 mentions)
Inject alum, by Thermo Fisher Scientific (2 mentions)
Lps e coli strain 055 b5, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Magainin 2, by Merck Group (1 mentions)
Saponin, by Fujifilm (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Digitonin, by Merck Group (1 mentions)
Gw4869, by Merck Group (1 mentions)
Bapta am, by Cayman Chemical (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
11 protocols using streptolysin o slo
1
Permeabilization of NIH 3T3 Cells by SLO
2
Plasma Membrane Damage Assay
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Adhered amoebae in a 24 well cell culture plate were washed with DMEM medium without Ca2+, and then replenish with complete DMEM medium (1.8 mM Ca2+) pre-warmed at 37 °C and PM damage was performed by adding streptolysin-O (SLO) (Sigma-Aldrich), the antimicrobial peptides Defensin and Magainin II (Sigma-Aldrich), and Triton X-100 (Sigma-Aldrich). After exposing the trophozoites to various incubation times, the viability of trophozoites was determined (see below), and cell-free supernatants were collected and assayed for the secreted aSMase activity as described above.
3
RBC Membrane Permeabilization for Imaging
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To selectively permeabilize the RBC membrane only, iRBC were treated with 20 U/ml streptolysin O (SLO; Sigma Aldrich Chemical Co, St. Louis, MO) in PBS for 6 min at room temperature, washed three times with ICM, and kept in ICM52 (link). To permeabilize RBC and PVM, iRBCs were treated with 0.01% saponin (Wako Pure Chemical Industries, Ltd, Japan) in PBS for 10 min at room temperature, washed three times with PBS, and kept in ICM53 (link). SLO- or saponin-treated iRBCs were transferred to hydrophilic-coated dish and kept for 30 min to let the iRBCs adhere to the glass dish bottom. The iRBCs were washed three times for 10 min each with Ca2+ free Tyrode’s buffer (140 mM NaCl, 10 mM glucose, 10 mM HEPES, 4 mM KCl, 1 mM MgCl2, pH 7.4) to remove Ca2+ from the extracellular medium. Finally, Ca2+ free Tyrode’s buffer was used for time lapse imaging.
4
Delivery of mMTRIPs into A549 Cells
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mMTRIPs were sequentially delivered into A549 cells using reversible membrane permeabilization. Briefly, 4U/ml Streptolysin O (SLO) (Sigma) were first reduced using 7.5mM Tris (2-carboxyethyl) phosphine (TCEP) (Pierce) for 1 hr at 37°C. Cells were rinsed using PBS (−Ca2+ −Mg2+) (Thermo) and then incubated with delivery medium containing 0.4U/ml SLO and probes at 15 nM in OptiMEM (Gibco) for 10 min at 37°C. The delivery medium was then removed and replaced with DMEM for recovery for 1h. 1x or 3x molar excess of the appropriate input gate was subsequently delivered using SLO. Cells were fixed using 4% paraformaldehyde and nuclei were stained with DAPI and mounted for imaging.
5
Macrophage Cytokine Responses to Uropathogenic E. coli
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BMDMs were incubated with UTI89, CFT073, or MG1655 for 2 hours, then treated with 100 μg/ml gentamicin containing medium for 1 hour and finally incubated with 10 μg/ml gentamicin containing medium for 5 hours (8-hr samples) or 21 hours (24-hr samples) at which point the supernatant was collected for ELISAs. IL-1β DuoSet ELISA kit (R&D systems, Minneapolis, MN), TNFα DuoSet ELISA kit (R&D systems) and Caspase-1 (mouse) matched pair detection set (Adipogen, San Diego, CA) were used according to manufacturer instructions. Where indicated, the following inhibitors were used throughout the experiment: the Cathepsin B inhibitor CA-074 Me (Enzo Life Sciences, Inc., Farmingdale, NY) was used at 10 μM, the caspase-1 inhibitor Z WEHD-FMK (R&D systems) was used at 10 μM, and the VPS34 inhibitor 3-MA (Calbiochem, Millipore, Billerica, MA) was used at 2, 5 and 10 mM. Alum- and Silica-treated samples were pretreated with 100 ng/ml LPS(E. coli strain 055:B5, Sigma) for 2 hours, and then treated with 200 μg/ml of Inject Alum (Thermo Scientific, Rockford, IL) or 100 μg/ml of Silica (Thermo Scientific) in fresh media for 6 hours. Streptolysin O (SLO) (Sigma) was used at 10 μg/ml with 10 mM DTT for 5 min, and then the medium was changed and collected 6 hours later.
6
Lysosomal Calcium Signaling Assay
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Chemical reagents were used at the following concentrations, unless indicated otherwise: 1 mM l -leucyl-l -leucine O-methyl ester (LLOMe; Bachem, 4000725); 200 µM glycyl-l -phenylalanine 2-naphtylamide (GPN; Abcam, ab145914); 1500 U/ml streptolysin O (SLO; Sigma-Aldrich, S5265); 250 µM digitonin (Sigma-Aldrich, D141); 5 µM ionomycin (Sigma-Aldrich, I0634); 100 µM BAPTA-AM (Cayman Chemical, 15551); 75 nM LysoTrackerTM Red DND-99 (Thermo Fisher Scientific; L7528); 1 µg/ml doxycycline (Sigma-Aldrich;); and 10 µM GW4869 (Sigma-Aldrich, D1692). GW4869 was stored at −80 °C as a 2 mM stock suspension in dimethyl sulfoxide (DMSO). Just before use, the suspension was solubilized by the addition of 5% methane sulfonic acid as described previously ref. 42 (link).
7
Macrophage Cytokine Responses to Uropathogenic E. coli
8
ADSC Reprogramming using ESC Extract
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The fourth-passaged ADSCs were isolated by trypsinization and washed in cold Dulbecco’s phosphate-buffered saline (DPBS) three times. Then, 5×105 cells were resuspended in 500 μL ice-cold DPBS and incubated at 37°C for 2 min. Then, 500 ng/mL streptolysin O (SLO, Sigma) was added, and the cell suspension was incubated for 50 min in a 37°C water bath with occasional agitation. Afterward, the tube was placed on ice, 500 μL cold DPBS was added, and the cells were centrifuged at 1000 rpm for 5 min at 4°C.
The permeabilized ADSCs were suspended in 500 μL of the ESC extract with an ATP-regenerating system (1 mM ATP, 10 mM creatine phosphate, 25 μg/mL creatine kinase, 100 μM GTP, and 1 mM of each nucleotide tri-phosphate; all from Sigma) and incubated at 37°C with occasional agitation for 1 h. Then, the content of the tube was transferred to culture plates with a 2 mM CaCl2-containing growth medium to reseal the plasma membranes. The next day, the culture medium was renewed, and the reprogrammed cells were cultured for one or two additional weeks (Ext-one week ADSCs and Ext-two weeks ADSCs). The ADSCs, which underwent the same procedure except for incubation in the ADSC extract, were used as the control cells (Cont-ADSCs).
The permeabilized ADSCs were suspended in 500 μL of the ESC extract with an ATP-regenerating system (1 mM ATP, 10 mM creatine phosphate, 25 μg/mL creatine kinase, 100 μM GTP, and 1 mM of each nucleotide tri-phosphate; all from Sigma) and incubated at 37°C with occasional agitation for 1 h. Then, the content of the tube was transferred to culture plates with a 2 mM CaCl2-containing growth medium to reseal the plasma membranes. The next day, the culture medium was renewed, and the reprogrammed cells were cultured for one or two additional weeks (Ext-one week ADSCs and Ext-two weeks ADSCs). The ADSCs, which underwent the same procedure except for incubation in the ADSC extract, were used as the control cells (Cont-ADSCs).
9
Propidium Iodide Staining for Cell Damage
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Cell damage was evaluated using propidium iodide (PI) staining, as done before (Encarnação et al, 2016 (link)). Cells were washed with HBSS, trypsinized, and 200 ng/ml streptolysin O (SLO) (Sigma‐Aldrich, Steinheim, Germany) was added in HBSS (Ca2+ and Mg2+ free) containing DTT (10 mM). The SLO‐containing HBSS was removed and replaced by fresh HBSS (Ca2+ free) or EBSS (Ca2+ complete) for 10 min at 37°C. Cells were then stained using 50 μg/ml propidium iodide (PI) with RNase A (250 μg/ml), incubated at 37°C for 30 min, and analyzed by FACS Calibur (BD Biosciences, Franklin Lakes, USA) and FlowJo software.
10
Delivery of mMTRIPs into A549 Cells
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