A375 cells stably expressing enCas12a were transduced with 6 triple knockout arrays, 3 single knockout constructs, and one empty control vector. Two days after transduction, cells were selected with puromycin (1μg/mL), and selected on puromycin for 7 days. Cells were visualized by flow cytometry on the BDAccuri C6 Sampler. To prepare samples for visualization, cells were stained with FITC anti-human CD47 (Biolegend # 323106), PE anti-human CD63 (Biolegend #353004) and APC anti-human β2-microglobulin (Biolegend #316312) antibodies, diluted 1:100 in flow buffer (PBS, 2% FBS, 5μM EDTA), incubated for 30 min on ice, washed with flow buffer twice to remove residual antibody, and resuspended in flow buffer. Flow cytometry data were analyzed using FlowJo (v10). Gates were set such that ~1% of cells score as knockout in the control condition. Compensation was applied using single stained empty vector control and triple stained empty vector control cell populations. Zero, single, double, and triple knockout populations were quantified using boolean gating in FlowJo.
Pe anti human cd63
Manufactured by BioLegend
Sourced in United States
PE anti-human CD63 is a fluorescent-labeled antibody used for the detection and analysis of CD63 expression on human cells. CD63 is a glycoprotein that is involved in the regulation of cell adhesion, motility, and signaling. The PE (Phycoerythrin) fluorescent label allows for the visualization and quantification of CD63-positive cells using flow cytometry or other fluorescence-based detection methods.
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Lab products found in correlation
Apc anti human β2 microglobulin, by BioLegend (2 mentions)
Fitc anti human cd47, by BioLegend (2 mentions)
C6 sampler, by BD (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Pe anti human cd9, by BioLegend (1 mentions)
Anti human igg pe, by BioLegend (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride, by Thermo Fisher Scientific (1 mentions)
Apc anti human cd81, by BioLegend (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Mouse anti gapdh, by Proteintech (1 mentions)
Minimum essential medium (mem), by Corning (1 mentions)
Ab108252, by Abcam (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Fitc anti human cd15, by BioLegend (1 mentions)
Pe cy7 anti human cxcr4, by BioLegend (1 mentions)
5 protocols using pe anti human cd63
1
Multiplexed Knockout Screening in A375 Cells
2
Exosomal Surface Protein Analysis
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An exosomal volume corresponding to 20 μg of protein was used for on-bead flow cytometry using anti-CD63 antibody-coated Dynabeads (ThermoFisher). Briefly, for each sample, 20 μL of the beads was washed with isolation buffer (PBS + 0.1% BSA, from MilliporeSigma) which was sterile filtered with a 0.22 μm syringe filter, mixed with exosome samples, and rocked overnight at 4 °C. The samples were then incubated with PE-anti-human CD63 (RRID: AB_10896786), PE-anti-human CD9 (RRID: AB_2075893), or PE-anti-human CD81 antibodies (RRID: AB_10642024, all antibodies from BioLegend, San Diego, CA, USA). Samples incubated with PE-anti-human IgG (RRID: AB_326435, BioLegend) were used as isotype controls, whereas samples not incubated with any antibodies were used as unstained controls. Samples were analyzed on a BD Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the data processed using FlowJo software 10.9.0 (BD Biosciences).
3
Antibodies for EV and Protein Detection
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DMEM, FBS, and penicillin-streptomycin were obtained from Gibco. MEM was purchased from Corning. Lipofectamine 2000 and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Thermo Fisher Scientific. MG132 was purchased from Selleck. All the pools of siRNA were obtained from the RiboBio Company (Guangzhou, China). The antibodies used for flow cytometry included PE-conjugated antibodies against CD63 (PE-anti-human CD63) (353003, BioLegend), APC-anti-human CD9 (312107, BioLegend), and APC-anti-human CD81 (349509, BioLegend). The antibodies used for Western blot included rabbit-anti-ubiquitin (10201-2-AP, Proteintech), mouse-anti-RFP-Tag (T0055, Affinity), rabbit-anti-ACE2 (ab108252, Abcam), mouse-anti-calnexin (66903-1-IG, Proteintech), rabbit-anti-CD63 (25682-1-AP, Proteintech), rabbit-anti-HA (51064-2-AP, Proteintech), rabbit-anti-GFP-Tag (50430-2-AP, Proteintech), mouse-anti-GAPDH (60004-1-Ig, Proteintech), and rabbit-anti-PSMD12 (39119, Signalway Antibody). The secondary antibodies used for developing targeted proteins in Western blotting assays included IRDye 680RD Goat anti-Mouse (926-68070) and IRDye 800CW Goat anti-Rabbit (926-32211).
4
Multiplexed Knockout Screening in A375 Cells
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A375 cells stably expressing enCas12a were transduced with 6 triple knockout arrays, 3 single knockout constructs, and one empty control vector. Two days after transduction, cells were selected with puromycin (1μg/mL), and selected on puromycin for 7 days. Cells were visualized by flow cytometry on the BDAccuri C6 Sampler. To prepare samples for visualization, cells were stained with FITC anti-human CD47 (Biolegend # 323106), PE anti-human CD63 (Biolegend #353004) and APC anti-human β2-microglobulin (Biolegend #316312) antibodies, diluted 1:100 in flow buffer (PBS, 2% FBS, 5μM EDTA), incubated for 30 min on ice, washed with flow buffer twice to remove residual antibody, and resuspended in flow buffer. Flow cytometry data were analyzed using FlowJo (v10). Gates were set such that ~1% of cells score as knockout in the control condition. Compensation was applied using single stained empty vector control and triple stained empty vector control cell populations. Zero, single, double, and triple knockout populations were quantified using boolean gating in FlowJo.
5
Neutrophil Degranulation Analysis in Psoriasis
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Degranulation of neutrophils was assessed by monitoring the cell surface expression of CD63. Blood samples obtained from healthy controls and psoriasis patients were collected and red blood cells were removed using Red Blood Lysing Buffer (FXP001, 4 A Biotech Co., Ltd). Then cells were labeled with FITC anti-human CD15 (301904, 1:100, BioLegend), PE-Cy7 anti-human CXCR4 (306514, 1:100, BioLegend) and PE anti-human CD63 (353004, 1:100, BioLegend) at 4 °C for 30 min. After three washes, cells were resuspended in PBS and analyzed using flow cytometry (649225, BD LSRFortessa Cell Analyzer), Background fluorescence levels were determined by Fluorescence Minus One (FMO).
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