Lung tissues were diced and subsequently digested for 45 min at 37 °C with 5 mg/ml LiberaseTM (Sigma, St Louis, MO, USA) and 10 mg/ml DNase (Roche, Basel, Switzerland). Single‐cell suspensions were stained and analyzed using a FACS Canto II cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Antibodies were purchased from BioLegend unless otherwise listed. For monocytes and neutrophils, the following antibodies were used: Ly6G (1A8), CD45 (30‐F11; eBioscience, San Diego, CA, USA), CD11c (HL3; BD, San Jose, CA, USA), CD11b (M1/70; BD), Ly6C (AL‐21; BD), and IL1RL1 (DIH9). For ILC staining, the following antibodies were used: CD3 (145‐2C11), CD19 (6D5), Gr1 (RB6‐8C5), B220 (RA3‐6B2), Ter119 (TER‐119), FcaR1 (MAR‐1), CD49b (DX5; eBioscience), Il1rl1 (DIH9), CD90 (30‐H12), and CD45 (30‐F11). A Fixable Viability Dye kit (eBioscience) was used to exclude dead cells. Data were analyzed using FlowJo software (Tree Star Inc, Ashland, OR, USA).
Fixable viability dye kit
Manufactured by Thermo Fisher Scientific
The Fixable Viability Dye kit is a fluorescent labeling reagent used to identify viable and non-viable cells in flow cytometry analysis. The kit provides a simple and reliable method to distinguish live and dead cells within a sample.
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Lab products found in correlation
Liberase, by Merck Group (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Cd11b m1 70, by BD (1 mentions)
Cd11c hl3, by BD (1 mentions)
Ly6c al 21, by BD (1 mentions)
Cd49b dx5, by Thermo Fisher Scientific (1 mentions)
Cd45 30 f11, by Thermo Fisher Scientific (1 mentions)
Dnase, by Roche (1 mentions)
Fc blocker cd16 cd32, by Thermo Fisher Scientific (1 mentions)
Sh800 cell sorter, by Sony (1 mentions)
2 protocols using fixable viability dye kit
1
Lung Immune Cell Isolation and Analysis
2
Multiplexed Flow Cytometric Analysis of PBMCs
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After PBMCs were thawed, the Fixable Viability Dye Kit (eBioscience, San Diego, CA) was used to assess cell viability by FACS analysis (>90% in all samples). 500,000 viable singlet-events were sorted per sample using a Sony SH800 Cell Sorter (Sony Biotechnology, San Jose, CA). The sorted cells were incubated with Fc blocker (CD16/CD32, eBioscience) for 10 min, after which each sample was incubated with TotalSeq Hashtag antibody tags to enable multiplexing and subsequent deconvoluting. After Hashtag staining, the cells were pooled into four pools of five samples, each sample contributing equally to their respective pool. The cells were counted manually by light microscopy and Neubauer chambers. Each pooled sample was then incubated for 30 min with our TotalSeq oligo-conjugated antibody panel for later surface protein marker quantification. An overview of all antibodies that were used in the study is shown in Supplementary file 2 .
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