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Lyse fix and perm 3 buffers

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Lyse/Fix and Perm III buffers are laboratory reagents used for cell preparation and analysis. Lyse/Fix buffer is used to lyse and fix cells, while Perm III buffer is used for cell permeabilization. These buffers are designed to prepare cells for flow cytometry analysis, but their specific use and applications may vary depending on the research or clinical requirements.

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Lab products found in correlation

Lsrfortessa x 20, by BD (2 mentions) Foxp3 fixation permeabilisation concentrate and diluent, by Thermo Fisher Scientific (1 mentions) Cytofix cytoperm, by BD (1 mentions) Facs fortessa, by BD (1 mentions) Facs lsr 2, by BD (1 mentions) Annexin 5, by BD (1 mentions)

4 protocols using lyse fix and perm 3 buffers

1

Multiparametric Immune Cell Analysis

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PBMCs were rapidly thawed in medium heated to 37°C and kept overnight at 4°C. Cells were then immunostained during 30 min at 4°C with the appropriate monoclonal antibodies detailed in Supplementary file 1. Intracellular staining of transcription factors and cytotoxic molecules was performed with Foxp3 Fixation/Permeabilisation concentrate and diluent (eBioscience). Intracellular staining of cytokines and chemokines was performed with Cytofix/Cytoperm (BD Biosciences). Intracellular staining of phosphorylated proteins was performed with Lyse/Fix and Perm III buffers (BD Biosciences). Phosphorylated proteins were then stained during 40 min at RT. Kynurenine uptake was measured as previously described (Sinclair et al., 2018 (link)). Briefly, PBMCs were stained for surface markers for NK cell identification and resuspended in phosphate-buffered saline (PBS). Baseline fluorescence in the BV421 channel was recorded for 30 s, and kynurenine (200 µM final concentration) was added before a further 4 min acquisition. The assay was run at 37°C. Flow cytometric analysis was performed on LSR Fortessa 5L (Becton-Dickinson). Fluorescence Minus One controls were used to set the gates, and data were analysed with FlowJo 10.5.0 software (Tree Star). Gating strategy is presented is Figure 1—figure supplement 2.
Marotel M., Villard M., Drouillard A., Tout I., Besson L., Allatif O., Pujol M., Rocca Y., Ainouze M., Roblot G., Viel S., Gomez M., Loustaud V., Alain S., Durantel D., Walzer T., Hasan U, & Marçais A. (2021). Peripheral natural killer cells in chronic hepatitis B patients display multiple molecular features of T cell exhaustion. eLife, 10, e60095.
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2

Enrichment and Analysis of CD4+ T Cells

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Tissues were mechanically dissociated to single cell suspensions and enriched for CD4+ T cells following an incubation with cocktail of antibodies (listed in Key Resource Table) to deplete non-CD4+ T cell lineages. Antibody-bound cells were removed by magnetic bead depletion using BioMag Goat anti-Rat IgG beads. Enriched CD4+T cells were stained with NP311–325:1-Ab or GP66–77:1-Ab, conjugated with streptavidin-PE tetramer for 1h at room temperature (Moon et al., 2011 (link)). Cells were stained using indicated antibodies (Key Resource Table) and intracellular proteins were detected using Foxp3 staining kit according to manufacturer’s protocol. Cytokine intracellular staining experiment was performed as previously described (Lüthje et al., 2012 (link)). For pSTAT5 detection, T cells were stimulated in round bottom plates in the presence of 100U/mL murine recombinant IL-2 (DNAX Research Institute) for 25 min at 37 degrees. Intracellular staining of pSTAT5 fixation and permeabilization with Lyse/Fix and Perm III buffers (BD Biosciences) as previously described (Viel et al., 2016 (link)). Cytometry data was acquired using a LSR Fortessa x-20 (BD Biosciences).
Sheikh A.A., Cooper L., Feng M., Souza-Fonseca-Guimaraes F., Lafouresse F., Duckworth B.C., Huntington N.D., Moon J.J., Pellegrini M., Nutt S.L., Belz G.T., Good-Jacobson K.L, & Groom J.R. (2019). Context-Dependent Role for T-bet in T Follicular Helper Differentiation and Germinal Center Function following Viral Infection. Cell reports, 28(7), 1758-1772.e4.
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3

Enrichment and Analysis of CD4+ T Cells

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Tissues were mechanically dissociated to single cell suspensions and enriched for CD4+ T cells following an incubation with cocktail of antibodies (listed in Key Resource Table) to deplete non-CD4+ T cell lineages. Antibody-bound cells were removed by magnetic bead depletion using BioMag Goat anti-Rat IgG beads. Enriched CD4+T cells were stained with NP311–325:1-Ab or GP66–77:1-Ab, conjugated with streptavidin-PE tetramer for 1h at room temperature (Moon et al., 2011 (link)). Cells were stained using indicated antibodies (Key Resource Table) and intracellular proteins were detected using Foxp3 staining kit according to manufacturer’s protocol. Cytokine intracellular staining experiment was performed as previously described (Lüthje et al., 2012 (link)). For pSTAT5 detection, T cells were stimulated in round bottom plates in the presence of 100U/mL murine recombinant IL-2 (DNAX Research Institute) for 25 min at 37 degrees. Intracellular staining of pSTAT5 fixation and permeabilization with Lyse/Fix and Perm III buffers (BD Biosciences) as previously described (Viel et al., 2016 (link)). Cytometry data was acquired using a LSR Fortessa x-20 (BD Biosciences).
Sheikh A.A., Cooper L., Feng M., Souza-Fonseca-Guimaraes F., Lafouresse F., Duckworth B.C., Huntington N.D., Moon J.J., Pellegrini M., Nutt S.L., Belz G.T., Good-Jacobson K.L, & Groom J.R. (2019). Context-Dependent Role for T-bet in T Follicular Helper Differentiation and Germinal Center Function following Viral Infection. Cell reports, 28(7), 1758-1772.e4.
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4

Multiparametric Flow Cytometry Analysis of Immune Cells

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Single-cell suspensions of mouse spleen, liver, blood and BM were obtained and stained. Intracellular stainings for TFs were performed using Foxp3 kit (eBioscience). Cell viability was measured using Annexin-V (BD Biosciences)/live-dead fixable (eBiosciences) staining. Lyse/Fix and PermIII buffers (BD Biosciences) were used for intracellular staining of phosphorylated proteins. Flow cytometry was carried out on a FACS LSR II, or a FACS Fortessa (Becton–Dickinson). Data were analysed using FlowJo (Treestar). Antibodies were purchased from eBioscience, BD biosciences, R&D Systems, Abcam, Beckman–Coulter, Miltenyi or Biolegend. We used the following antibodies (Supplementary Table 1): anti-CD3 (clone 145-2C11), anti-CD19 (clone 1D3), anti-NK1.1 (clone PK136), anti-NKP46 (clone 29A1.4), anti-CD11B (clone M1/70), anti-CD27 (clone LG.7F9), anti-CD49A (clone HA31/8), anti-CD49B (clone DX5), anti-CD122 (clone SH4), anti-CD127 (clone A7R34), anti-CD62L (clone MEL-14), anti-Ly49A (clone YE1/48.10.6), anti-Ly49D (clone 4E5), anti-Ly49G2 (clone 4D11), anti-CD226 (clone 10E5), anti-Tbet (clone 4B10), anti-EOMES (clone Dan11mag), anti-Granzyme A (clone 3G8.5), anti-KI67 (clone SolA15), anti-KLRG1 (clone 2F1), anti-STAT4 (clone 38/p-Stat4), anti-HA (clone 6 E2), anti-IFNγ (clone Dan11mag).
Zhang J., Le Gras S., Pouxvielh K., Faure F., Fallone L., Kern N., Moreews M., Mathieu A.L., Schneider R., Marliac Q., Jung M., Berton A., Hayek S., Vidalain P.O., Marçais A., Dodard G., Dejean A., Brossay L., Ghavi-Helm Y, & Walzer T. (2021). Sequential actions of EOMES and T-BET promote stepwise maturation of natural killer cells. Nature Communications, 12, 5446.
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