For staining of macrophages, splenic immune cells and lamina propria lymphocytes, cells were preincubated with PBS containing 0.5% BSA and FCγIII/IIR antibody to block nonspecific binding. The Cell Trace CFSE Cell Proliferation Kit (Invitrogen) and the 7-AAD Viability Solution (eBioscience) was used according to the manufacturer's instruction. Cells were stained with corresponding antibodies (supplemental methods) and analyzed using a FACSCantoTM II flow cytometer (BD Biosciences) or a FACS Fortessa (Beckton Dickinson) and data were calculated with FACSDiva software (BD Biosciences).
7 aad viability solution
Manufactured by Thermo Fisher Scientific
The 7-AAD Viability Solution is a fluorescent dye used to detect dead cells in flow cytometry analysis. It binds to the DNA of cells with compromised membrane integrity, allowing for the identification and quantification of non-viable cells within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Celltrace cfse cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Facs fortessa, by BD (1 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
2 protocols using 7 aad viability solution
1
Multiparametric Immune Cell Profiling
2
Flow Cytometry Analysis of T Cell Viability
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A Thermo Fisher Attune Nxt flow cytometer was used for assessment of viability and efficiency metrics. 200 μL of cultured cells were pelleted (500 x g, 5 min) and resuspended in Dulbecco's phosphate-buffered saline (DPBS) from Fisher Scientific (#14190250) with 7-AAD viability solution from eBiosciences (#00-6993-50). The cells were then analyzed on a volumetric read using the Attune Nxt autosampler. Total cells were gated in the forward scatter (FSC) and side scatter (SSC) dot plots. Viable cells (7-AAD -) were then gated to determine delivery efficiency via expression of the fluorescent reporters. This gating strategy is shown in Supplementary Figure 1. Total cell counts and yields were calculated from an applied dilution factor based on total volume of the cell culture (7.5X per 1 mL). Naïve T cell marker staining was performed with APC/Cy7 mouse anti-human CD45RA (#304128) and BV510 mouse anti-human CD45RO (#304232) antibodies from Biolegend.
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