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.
Fitc anti human cd47
Manufactured by BioLegend
Sourced in United States
The FITC anti-human CD47 is a fluorescently labeled antibody that binds to the CD47 antigen expressed on the surface of human cells. CD47 is a membrane protein involved in various cellular processes. This antibody can be used for the detection and analysis of CD47-expressing cells in research applications.
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Lab products found in correlation
Apc anti human β2 microglobulin, by BioLegend (2 mentions)
Pe anti human cd63, by BioLegend (2 mentions)
C6 sampler, by BD (2 mentions)
Chaetocin, by Merck Group (1 mentions)
Anti c jun, by Abcam (1 mentions)
Sp600125, by Merck Group (1 mentions)
Phospho jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Anti cd47, by Abcam (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Z vad fmk, by Enzo Life Sciences (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
3 protocols using fitc anti human cd47
1
Multiplexed Knockout Screening in A375 Cells
2
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.
3
Apoptosis Signaling Pathway Inhibitors
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Chaetocin (#C9492, Sigma-Aldrich, St Louis, MO, United States), z-VAD-fmk (#ALX-260–020, Enzo, New York, NY, United States), N-acetyl-L-cysteine (NAC) (#A7250, Sigma-Aldrich) and SP600125 (#S5567, Sigma-Aldrich) were dissolved in DMSO to form the 10mM, 50mM, 0.5M and 20 mM solution respectively. These reagents were stored at −20°C. Primary antibodies against PARP (#9542), caspase-3 (#9662), cleaved-caspase-3 (#9661), caspase-8 (#9746), caspase-9 (#9508), BCL-2 (#15071), BCL-XL (#2764), MCL-1 (#4572), XIAP (#14334), JNK (#9252) and phospho-JNK (Thr183/Tyr185) (#4668) were purchased from Cell Signaling Technology (Beverly, MA, United States). Anti-c-Jun (#ab131497), anti-Ser63 phosphor-c-Jun (#ab28807), anti-CD47 (#ab9089) anti-α-tubulin (#ab233661) and β-actin (#ab8226) were purchased from Abcam (Cambridge, MA, United States), FITC anti-human CD47 (#323106) was purchased from Biolegend (San Diego, CA, United States). Anti-mouse immunoglobulin G (#B900620) and anti-rabbit immunoglobulin G (#B900610) horseradish peroxidase-conjugated secondary antibodies were purchased from Proteintech Group (Chicago, IL, United States).
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