Anti-CD15-APC (Thermo Fisher Scientific, Whaltman, MA, USA; Cat. No. 17-0158-42), anti-CD14-FITC (Thermo Fisher Scientific, Whaltman, MA, USA; Cat. No. 11-0149-42) and anti-CD11b-Pacific blue (BioLegend, San Diego, CA, USA; Cat. No. 101224) were incubated with 1 × 106 K562 cells (vehicle or NAD treated) at 1:100 ratio. Cells were pre-incubated with anti-Fc receptor antibody (Thermo Fisher Scientific, Whaltman, MA, USA Cat. No. 14-9161-73) at 1:20 ratio to block Fc receptor before staining. Zombie red staining (BioLegend, San Diego, CA, USA Cat. No. 423109) was used as cell viability dye during FACS analysis. Cells were fixed using 2% PFA (Sigma Aldrich, St. Louis, MO, USA; Cat. No. 158127) before performing FACS analysis. Cell acquisition and analysis were performed on BD LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA) using BD FACSDivaTM software (BD Bioscience, Franklin Lakes, NJ, USA). Analysis was performed using Flowjo software (Flowjo LLC, Ashland, OR, USA).
Zombie red staining
Manufactured by BioLegend
Sourced in United States
Zombie red staining is a fluorescent dye used for detecting cell death in flow cytometry applications. It binds to dead cells, allowing for their identification and quantification within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (2 mentions)
Pacific blue anti cd11b, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Anti cd14 fitc, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Iscove modified dulbecco media (imdm), by Lonza (1 mentions)
Cd4 bv510 sk3, by BD (1 mentions)
3 laser aurora, by Cytek (1 mentions)
Cd8 apc h7 sk1, by BD (1 mentions)
L glutamine, by Lonza (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
3 protocols using zombie red staining
1
Flow Cytometry Analysis of K562 Cells
2
Analyzing IFN-γ and TNF-α in PBMCs
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PBMCs from healthy CMV-seropositive donors that express HLA-DPB1*01:01 and/or HLA-DPB1*05:01 were incubated (37°C + 5% CO2) with 1 µg/mL peptide in IMDM (Lonza) with 10% fetal bovine serum (Thermo Fisher Scientific), 200 mM L-glutamine (Lonza), and 10,000 U/ml penicillin/streptomycin (Lonza). Half of medium was refreshed daily, and after 10 d of culturing, the cells were restimulated with the same peptide. After 1-h incubation, 5 µg/mL Brefeldin A (Sigma-Aldrich) was added and incubated for an additional 15 h. The reaction was stopped by washing in PBS, followed by Zombie-red staining (BioLegend). After incubation for 20 min at room temperature (RT), cells were fixated and permeabilized using the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), according to the manufacturer’s protocol. Cells were incubated for 30 min at RT with an antibody mix containing Brilliant Violet Staining Buffer Plus (BD Biosciences), CD3-PE-Texas Red (7D6; Invitrogen), CD8-APC-H7 (SK1; BD Biosciences), CD4-BV510 (SK3; BD Biosciences), IFNγ-BV711 (B27; BD Biosciences), and TNFα-BV421 (Mab11; BD Biosciences). After incubation, cells were washed and resuspended in PBS containing 400 mg albumin for measurement on a 3-laser Aurora (Cytek Biosciences). Data were analyzed using OMIQ (https://www.omiq.ai/ ).
3
Immunophenotyping of NAD-Treated K562 Cells
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Anti-CD15-APC (Thermal Fisher Scientific, Cat. No. 17-0158-42), anti-CD14-FITC (Thermal Fisher Scientific, and anti-CD11b-Pacific blue (BioLegend, Cat. No. 101224) were incubated with 1x10 6 K562 cells (vehicle or NAD treated) at 1:100 ratio. Cells were preincubated with anti-Fc receptor antibody (Thermal Fisher Scientific, Cat. No. 14-9161-73) at 1:20 ratio to block Fc receptor before staining. Zombie red staining (BioLegend, Cat. No. 423109) was used as cell viability dye during FACS analysis. Cells were fixed using 2% PFA (Sigma, Cat. No. 158127) before performing FACS analysis. Cell acquisition and analysis were performed on BD LSRFortessa (BD biosciences, CA, USA) using BD FACSDiva TM software (BD Bioscience).
Analysis was performed using Flowjo software (Flowjo LLC, USA).
Analysis was performed using Flowjo software (Flowjo LLC, USA).
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