Freshly isolated B cells were treated with Human Fc Receptor Binding Inhibitor (eBioscience, San Diego, CA) to block the Fc receptor prior to staining. Cells were then stained with FITC-conjugated anti-CD27, PE-conjugated anti-CD19, PE-Cy7-conjugated anti-CD20, BV421-conjugated anti-CD38 and APC-conjugated anti-CD3 (Biolegend, San Diego, CA). The LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Life Technologies, Carlsbad, CA) was used to determine the viability of cells. Single plasmablasts (CD3−CD19+CD20lo/−CD27hiCD38hi) were sorted into 96-well plates with 4 μl of cell lysis buffer containing 10 mM DTT (Invitrogen, Carlsbad, CA) and RNase Inhibitor (Eppendorf) in each well (13 (link)). Cell sorting was performed using a BD InfluxTM cell sorter.
Rnase inhibitor
Manufactured by Eppendorf
The RNase Inhibitor is a laboratory reagent designed to prevent the degradation of RNA molecules by ribonuclease (RNase) enzymes. It functions by inhibiting the activity of RNase, thereby protecting the integrity and stability of RNA samples for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Human fc receptor binding inhibitor, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated anti cd27, by BioLegend (1 mentions)
Pe conjugated anti cd19, by BioLegend (1 mentions)
Pe cy7 conjugated anti cd20, by BioLegend (1 mentions)
Apc conjugated anti cd3, by BioLegend (1 mentions)
Influx cell sorter, by BD (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Flag tag polyclonal antibody, by Proteintech (1 mentions)
Rnase inhibitor, by ABclonal (1 mentions)
2 protocols using rnase inhibitor
1
Isolation and Characterization of Plasmablasts
2
In Vivo CLIP Assay Protocol
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The in vivo crosslinking and immunoprecipitation (CLIP) assay was performed according to the previous description with fine‐tuned adjustments.27 Shortly, cells cultured in a 10‐cm dish were harvested and cross‐linked under ultraviolet. Then IP procedure was conducted by Flag‐tag polyclonal antibody (catalog number: 20543‐1‐AP) or rabbit IgG polyclonal antibody (catalog number: 30000‐0‐AP) from Proteintech Group. Cell extracts were incubated with indicated antibodies overnight at 4°C followed by coincubation with 50 µl magnetic beads (MCE, catalog number: HY‐K0205) per pipe for 1 h. The next step was to clean the beads three times by washing buffer with 1% cocktail and 1 U/µl RNase inhibitor (Abclonal, catalog number: RK21401) and suspending in 120 µl elution buffer with 1% cocktail and 1 U/µl RNase inhibitor at 30°C for 15 min. Then another Eppendorf tube was used to collect the supernatant, and 5 µl 4.8 M NaCl with 1 U/µl RNase inhibitor was added followed by shaking overnight at 65°C. Proteinase K was used to digest the protein therein at 60°C for 1 h. Following RNA extraction and reverse transcription with random primers, PCR assays were performed using specially designed primers to amplify the skipped cassette as well as the flanking exons.
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