Cells were lysed on ice for 5 min employing a buffer containing 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 20 mM ß-glycerophosphate, 1 mM Na3VO4, 1% IGEPAL (Thermo Fisher Scientific), and 1x protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation (20.000g at 4°C for 5 min). Protein concentrations were determined using the Bradford assay.
Igepal
Manufactured by Thermo Fisher Scientific
Sourced in United States
IGEPAL is a non-ionic surfactant commonly used in laboratory applications. It is a versatile detergent that can be used in various biochemical and molecular biology procedures to solubilize proteins, disrupt cell membranes, and facilitate the extraction and purification of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Rnaseout, by Thermo Fisher Scientific (3 mentions)
Cd20 cy7apc, by BD (2 mentions)
Cd3 cy7pe, by BD (2 mentions)
Cd3 cd20 cd19, by Thermo Fisher Scientific (2 mentions)
Aqua live dead dye, by Thermo Fisher Scientific (2 mentions)
Cd27 apc, by Thermo Fisher Scientific (2 mentions)
Cd19 fitc, by BD (2 mentions)
Cd38 pe, by BD (2 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Rnase a, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Fastprep 24 classic, by MP Biomedicals (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Facsaria iiiu, by BD (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
6 protocols using igepal
1
Cell Lysis and Protein Extraction
2
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested in ice and washed with PBS. They were fixed in 70% ethanol for 30 min at −20°C. They were washed twice in PBS pelleted by centrifugation at 850 g for 5 min at 4°C. The cells were then resuspended in a solution containing 3.5 mM Tris–HCl pH 7.6 (Thermo Scientific), 10 mM NaCl (Thermo Scientific), 50 μg/ml propidium iodide (Sigma P4170), 0.1% IGEPAL (Thermo Scientific), 20 μg/ml RnaseA (Sigma), and water. The acquisition of stained cells was performed using an LSRII flow cytometer (BD Biosciences). The acquired data were analyzed using FlowJo software.
3
V5-tagged Protein Immunoprecipitation
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Cells were grown in YEPD to 1.1 x 107 cells /ml. 1.1 x 109 cells were collected and frozen. Cells were lysed with a FastPrep-24 Classic (MP Biomedicals, Speed 6.5, 60s, 10 cycles) with 200μl NP40 buffer [50mM Tris pH7.5, 150mM NaCl, 1% Np40 (IGEPAL) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific)]. Lysates were brought up to 1.5ml with NP40 buffer and were pelleted by centrifugation at 20,000g for 10 minutes at 4C. 1% of sample was taken for Input and boiled with 10μl of 3x SDS sample buffer, and the rest of the lysates were brought to 0.2% BSA. Clarified sample was added to 20μl of Anti-V5 agarose affinity gel antibody beads (Sigma) and incubated for 2 hours at 4C. Beads were washed 3 times with Np40 buffer with Protease inhibitor and 2 times in NP40 buffer without inhibitors. To elute, 30μl of 1.5x SDS sample buffer was added to the beads and samples were boiled for 5 minutes.
4
Isolation and Sorting of Memory B Cells
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Blood samples were donated by a healthy volunteer. PBMC were isolated and stained with the indicated cell-surface markers. Single memory B cells were sorted into 96-well PCR plates as described previously [25 (link)]. Briefly, peripheral blood mononuclear cells (PBMC) were isolated from blood and then stained with the indicated cell-surface markers. Memory B cells were gated as CD19+ IgM− CD27+. SA-APC A33+ and SA-PB A33+ double-positive memory B cells were sorted as single cells into 96-well PCR plates containing 20 μL/well of RT reaction buffer that included 5 μL of 5× First strand cDNA buffer, 0.5 μL of RNAseOut (Invitrogen, Carlsbad, CA, USA), 1.25 μL of DTT, 0.0625 μL of Igepal, and 13.25 μL of dH2O (Invitrogen, Carlsbad, CA, USA) The plates were briefly centrifuged and immediately stored at −80 ℃ until further processing. Cells were sorted on a BD FACS Aria IIIu, and the data were analyzed using FlowJo.
5
Single-cell sorting of plasmablasts
6
Single-cell sorting of plasmablasts
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PBMCs were freshly isolated from blood samples collected 7 days after PfSPZ immunization and stained for viability with Aqua Live/Dead dye(Invitrogen) followed by surface-staining for the following markers: CD20-Cy7APC (BD Bioscience), CD19-FITC (BD Bioscience), CD3-Cy7PE (BD Bioscience), CD38-PE (BD Bioscience), and CD27-APC (ThermoFisher). Plasmablasts were gated as live, CD3-CD20-CD19+CD27+CD38+ and were sorted as single cells into 96-well PCR plates containing 20 μl/well of RT reaction buffer that included 5 μl of 5× First strand cDNA buffer, 0.5 μl of RNAseOut (Invitrogen), 1.25 μl of DTT, 0.0625 μl of Igepal and 13.25 μl of dH2O (Invitrogen) as previously described46 (link).
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