Calu-3 cells were pretreated with lapatinib (10 μM) or DMSO for 1 hour at 37°C and infected with SARS-CoV-2 (MOI = 1) at 4°C. After 1-hour incubation, the temperature was shifted to 37°C to initiate infection. At 0.5 and 1 hour after temperature shift, cells were washed with PBS and incubated for 15 minutes at room temperature with Zombie Aqua live/dead fixable dye (BioLegend) and FcR Blocking Reagent (Miltenyi Biotec). Cells were stained for 20 minutes at 4°C with anti-NRP1–BV421, anti-ACE2–APC, and anti-ErbB2–Alexa Fluor 488 antibodies, or with their corresponding isotype controls. Unbound antibody was washed, and cells were fixed with 4% PFA for 1 hour at room temperature. Cell acquisition was performed on an Aurora Cytek spectral flow cytometer, and data were analyzed using FlowJo software (Tree Star).
Zombie aqua live dead fixable dye
Manufactured by BioLegend
Zombie Aqua live/dead fixable dye is a fluorescent staining reagent for detecting cell viability. It functions by binding to cellular proteins, enabling the identification of live and dead cells in a sample.
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Lab products found in correlation
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Fixation buffer, by BioLegend (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Compensation beads, by BD (1 mentions)
Lysis buffer, by BioLegend (1 mentions)
Collagenase p, by Merck Group (1 mentions)
Trustain fcx anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Dnase 1, by Merck Group (1 mentions)
2 protocols using zombie aqua live dead fixable dye
1
SARS-CoV-2 Infection Kinetics in Calu-3 Cells
2
Immune Cell Profiling of Tumor Samples
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Tumors with knockout of Mettl3, Mettl14, and control were collected from mice, weighted, mechanically diced, and then digested with 2 mg/ml collagenase P (Sigma‐Aldrich) and 50 μg/ml DNase I (Sigma‐Aldrich) at 37°C for 30 min. Then, these samples were filtered through 70‐μm cell strainers and washed by cell staining buffer (BioLegend). The red blood cells were lysed with lysis buffer (BioLegend, 420301). After counting viable cells and these cells were blocked with TruStain FcX (anti‐mouse CD16/32) antibody (BioLegend) and then incubated with Zombie Aqua Live/Dead fixable dye (BioLegend, 423102). Subsequently, specific antibodies recognized cell surface markers were stained. The intracellular staining procedures followed by the BioLegend protocol as recommended. Briefly, cells were fixed with fixation buffer (BioLegend, 420801), permeabilized, and stained with predetermined optimum combination of antibodies. Meanwhile, BD Compensation Beads (BD Biosciences, 552845) were used to optimize fluorescence compensation settings for multicolor flow cytometric analysis. Information about all the antibodies used in the flow cytometry analysis is provided below. CD45 (clone 30‐F11), CD3ε (clone 145‐2C11), CD4 (clone RM4‐5), CD8 (clone 53‐6.7), NK1.1(clone PK136), FoxP3 (clone MF‐14), granzyme B (clone QA16A02), and all the antibodies were purchased from BioLegend.
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