Spleens and lymph nodes were harvested into a tissue culture dish and teased apart into a single cell suspension. Cell suspension was passed through a cell 0.22 µM filter (EMD Millipore) and centrifuged (300–400 × g) at 4°C. Cell pellet was resuspended in Flow Cytometry Staining Buffer (BioLegend, San Diego, CA, USA) at the final concentration of 2 × 107 cells/mL. Cell surface and intracellular staining were performed according BioLegend protocol (BioLegend). Antibodies used for analysis are listed in Table S2 in Supplementary Material.
Flow cytometry staining buffer
Manufactured by BioLegend
Sourced in United States
Flow cytometry staining buffer is a solution designed to facilitate the staining of cells for analysis using flow cytometry. It is formulated to maintain the viability and integrity of cells during the staining process, enabling the effective labeling of cellular markers for subsequent detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Facsverse, by BD (1 mentions)
Cd3 apc cy7, by BD (1 mentions)
Cd56 bv421, by BD (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
Fixation and permeabilization buffer set, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4, by BioLegend (1 mentions)
Permeabilization buffer, by BioLegend (1 mentions)
Anti mouse cd3, by BioLegend (1 mentions)
Anti mouse cd8, by BioLegend (1 mentions)
Anti mouse cd25, by BioLegend (1 mentions)
Human pan monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Lsr 2 cytofluorimeter, by BD (1 mentions)
Calcium and magnesium, by Lonza (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Mouse igg1, by Thermo Fisher Scientific (1 mentions)
Fitc mouse anti human cd206, by BD (1 mentions)
Fitc mouse anti human cd86, by BD (1 mentions)
7 protocols using flow cytometry staining buffer
1
Isolation and Staining of Immune Cells
2
Single-Cell Isolation and Staining
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Spleens and lymph nodes were harvest into a tissue culture dish and tease it apart into a single cell suspension. Cell suspension was passed through a cell 0.22 μM filter and centrifuged (300–400 ×g) at 4 °C. Cell pellet was resuspended in Flow Cytometry Staining Buffer (Biolegend) at the final concentration of 2 × 107/ml. Cell surface and intracellular staining were performed according to Biolegend protocol (Biolegend). Antibodies used for analysis are listed in Supplementary Table 1 .
3
Multiparametric Flow Cytometry Analysis
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For phenotypical analysis, cells were washed with Flow Cytometry Staining Buffer (BioLegend) and stained with anti-Gr1, anti-Ly6G, anti-Ly6C, anti-CD11b, anti-CD11c, anti-F4/80, and anti-CD4 mAbs for 30 minutes at 4 °C. For the intracellular staining of CD4+ T cells, cultured CD4+ T cells stained with an anti-CD4 mAb were treated with the Intracellular Fixation & Permeabilization Buffer Set (BioLegend). These cells were analyzed on a FACS Verse (BD Bioscience) using FlowJo software (Tree Star).
4
Immunophenotyping of peripheral blood mononuclear cells
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Peripheral blood mononuclear cells were isolated from fresh blood and resuspended at 10 × 106/ml in flow cytometry staining buffer (#420201, BioLegend). Then, cells were incubated with fluorescent conjugated antibodies, including APC‐Cy7‐CD3 (#557757, BD Pharmingen) and BV421‐CD56 (#562751, BD Pharmingen), on ice for 20 min in the dark. To detect the expression of intracellular cytotoxic molecules, cells were fixed and permeabilized using Fixation and Permeabilization Buffer Set (#88‐8824, eBioscience), and then stained with GZMB antibody (#515406, BioLegend) or IgG1 κ (#400136, BioLegend) as an isotype control. After washing twice by centrifugation at 350 g for 5 min, cells were resuspended in 0.5 ml of staining buffer and harvested using CytoFlex S (Beckman Coulter). Data analysis was performed using FlowJo software (version 10).
5
Mouse Spleen Cell Immunophenotyping
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The antibodies used for flow cytometric analysis include FITC anti-mouse CD3, Pacific Blue anti-mouse CD4, PerCP anti-mouse CD8a, PE/Cyanine 7 anti-mouse CD19, APC anti-mouse CD25, PE anti-mouse FoxP3 and their corresponding isotype controls. All of them were obtained from Biolegend (San Diego, CA, USA).
The mouse spleen cells were suspended in RPMI 1640 (HyClone, Logan, UT, USA) medium with 10% fetal calf serum (Hangzhou Sijiqing Biological Engineering Materials) at a final concentration of 1×107cells/mL. Splenocytes were then suspended in a flow cytometry staining buffer (Biolegend), stained with surface antibodies (anti-mouse CD3, anti-mouse CD4, anti-mouse CD8, anti-mouse CD19 and anti-mouse CD25) for 30 min, incubated with permeabilization buffer (Biolegend) at room temperature for 1 hour and stained with the intracellular antibody (anti-mouse FoxP3) according to the manufacturer’s instructions. Flow cytometric analysis was performed using a flow cytometry machine (Beckmancount, Shanghai, China).
The mouse spleen cells were suspended in RPMI 1640 (HyClone, Logan, UT, USA) medium with 10% fetal calf serum (Hangzhou Sijiqing Biological Engineering Materials) at a final concentration of 1×107cells/mL. Splenocytes were then suspended in a flow cytometry staining buffer (Biolegend), stained with surface antibodies (anti-mouse CD3, anti-mouse CD4, anti-mouse CD8, anti-mouse CD19 and anti-mouse CD25) for 30 min, incubated with permeabilization buffer (Biolegend) at room temperature for 1 hour and stained with the intracellular antibody (anti-mouse FoxP3) according to the manufacturer’s instructions. Flow cytometric analysis was performed using a flow cytometry machine (Beckmancount, Shanghai, China).
6
Isolation and Characterization of Human Monocytes
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PBMCs were isolated from whole blood of healthy donors and diluted in PBS without calcium and magnesium (Lonza) 1× (ratio 1:1). Diluted samples were subjected to density gradient separation on Ficoll Paque Plus (ratio 1:1; GE Healthcare Life Sciences) in accordance with the manufacturer’s protocol. Monocytes were then isolated from PBMCs using the Human Pan Monocyte Isolation Kit (Miltenyi Biotec) following manufacturer’s instructions (magnetic bead negative selection). Monocytes were then either fixed in 3% paraformaldehyde or differentiated into monocyte-derived macrophages in culture. After 7 days in culture, macrophages were detached with trypsin and fixed in 3% paraformaldehyde. Fixed cells were permeabilized using 0.2% Triton/PBS (15 minutes incubation at room temperature), washed in 0.2% Tween/PBS 3×, and blocked in 5% horse serum/PBS for 20 minutes at room temperature. Samples were then incubated with the appropriate primary antibodies (Major Resources Table in the Data Supplement ) for 30 minutes at room temperature, washed in 0.2% Tween/PBS 3× and for nonconjugated primary antibodies incubated with secondary antibodies for 30 minutes at room temperature protecting from light. After 3 washes in 0.2% Tween/PBS, the samples were resuspended in flow cytometry staining buffer (Biolegend) and acquired using a LSRII Cytofluorimeter (BD Biosciences).
7
Detailed Flow Cytometry Protocol for Immune Cell Markers
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Cell surface markers were analyzed by flow cytometry. In brief, flow cytometry staining buffer (BioLegend, San Diego, CA) was added to the cells and blocking was performed by adding 2 μL of FcR blocking reagent per 10 6 cells (Miltenyi Biotec, Sweden), followed by gently vortexing the samples, and allowing the tubes to stand at room temperature for 15 min. The cells were then incubated with the conjugated antibodies, FITC mouse anti-human CD86 (BD Pharmingen) and FITC mouse anti-human CD206 (BD Pharmingen), and the isotypematched control antibody (mouse IgG1, Invitrogen), then gently vortexed, and incubated at room temperature for 30 min protected from light. The single cell suspensions were maintained by intermittent vortexing during the incubation. The cell pellets were resuspended in flow cytometry staining buffer for analysis using the BD LSRFortessa™ X-20 (BD Biosciences, San Jose, CA). Data were analysed using FCS Express™ v. 7.0 software (DeNovo Software, Pasadena, CA).
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