Complete peripheral blood was obtained from median cubital venipuncture and collected in sodium citrate anticoagulant tubes. Immediately, 0.1 mL of blood was transferred to polystyrene flow cytometer tubes, and 15 μL of the mix of antibodies FITC-CD15 (5 μL), PE-CD11b (5 μL) and BV450-CD66b (5 μL) were added; the tubes were incubated for 30 min at 4 °C in darkness. Afterward, 0.9 mL 1X of lysing and fixing solutions (BD FACS Lysing) were added to samples and incubated for 15 min at room temperature (RT) in darkness. Finally, 104 cells of the polymorphonuclear region were acquired on a BD FACS lyric flow cytometer (BD, San Diego, CA, USA); the acquired data were analyzed using FlowJo 10.0 v software (FlowJo LLC, Ashland, OR, USA).
Facs lysing
Manufactured by BD
Sourced in United States
BD FACS Lysing is a laboratory reagent used for the lysis of red blood cells in cell samples prior to flow cytometric analysis. It is designed to facilitate the preparation of samples for immunophenotyping and other flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (4 mentions)
Lsrfortessa, by BD (4 mentions)
Hemoton spec, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Cd3 fitc, by BD (2 mentions)
Cd8 percp, by BD (2 mentions)
Trucount tube, by BD (2 mentions)
Multitest 6 color tbnk reagent, by BD (2 mentions)
Cd28 pe, by BD (2 mentions)
Facscanto software, by BD (2 mentions)
Cd95 apc, by BD (2 mentions)
Brefeldin a, by Merck Group (2 mentions)
Facslyric flow cytometer, by BD (1 mentions)
Cellfix, by BD (1 mentions)
Horizon brilliant stain buffer, by BD (1 mentions)
Ccr2 apc, by Thermo Fisher Scientific (1 mentions)
Cd62l, by BD (1 mentions)
Tim 3, by BioLegend (1 mentions)
Cd57 apc clone nk 1, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
20 protocols using facs lysing
1
Quantification of Neutrophil Activation Markers
2
Immunosenescent Cell Analysis in Whole Blood
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UCLA Immune Assessment Core performed the analysis of immunosenescent cells for 144 whole blood samples, as described previously [71 (link)]. Briefly, Total CD3 + T-cells, CD4 + T-cells, CD8 + T-cells, CD19 + B-cells, and CD56 + /CD16 + NK-cells were enumerated in EDTA whole blood with the BD Multitest 6-color TBNK reagent and BD Trucount tubes following the manufacturer’s instructions, acquired on a BD FACSCanto II and analyzed with the BD FACSCanto Software. CD8 + T-cell sub setting was performed by staining 50 μl of EDTA whole blood with CD3 FITC, CD8 PerCP, CD28 PE, and CD95 APC (BD) for 10 min, followed by BD FACS Lysing used according to the manufacturer’s instructions. At least 10,000 lymphocyte events per sample were acquired and analyzed using DIVA 8.0 software on BD FACSCanto II.
3
Multicolor Flow Cytometry Staining
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All steps of the staining were performed at 4°C. Cell suspensions were first stained with viability dye LiveDead® (Thermofischer Scientific) for 15 min, then washed with PBS. Fc receptors were blocked using PBS supplemented with 5% macaque serum for 20 min. Cell suspensions were stained for 30 min with 90 µl antibody mix (Table 1 ) diluted in BD Horizon Brilliant® Stain Buffer (BD Biosciences, Franklin Lakes, NJ, USA). Cells were then washed twice in PBS and fixed using 150 µl BD Cellfix® (BD). Red blood cells were removed with 1 ml BD FACs Lysing® (BD) for 10 min at room temperature and washed twice with PBS. The data were normalized to the weight of the initial biopsy for the analysis of cell suspensions from tissues. Data from blood cells were normalized to the complete blood counts. The design of the cytometry panel and the rationale for gating strategies (Figure S3 in Supplementary Material) has been previously described (34 (link)).
4
Multicolor Flow Cytometry of Innate Immune Cells
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Directly conjugated antibodies were obtained from the following vendors: BD Biosciences (San Jose, CA): CD3 (FITC), CD14 (BV605), HLADR (PE-CF594), CD16 (APC-H7); R&D systems: CCR2 (APC); eBioscience (San Diego, CA): CX3CR1 (PE), CD62L (PerCP-eF710). Staining for innate cell surface markers was performed with 100 μl of fresh blood incubated 15 min with antibodies. The blood was then lysed with BD FACS lysing, resuspended with PBS 1X (BD Biosciences) and immediately analyzed by flow cytometry.
5
Standardized Flow Cytometry of Blood
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100 µL blood was stained for 30 min with 90 µL antibody mix in BD Horizon® staining buffer (BD) containing the panel of antibodies listed in Table S2
6
Peripheral Blood NK Cell Analysis
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For analysis of the activation/exhaustion/inhibition markers in the NK cells of the peripheral blood, venous blood was collected in EDTA-anticoagulated tubes, and staining was performed using the following antibodies: CD3 (BV605/Clone: SK7), CD19 (Horizon V500/Clone: HIB19), CD16 (APC-Cy7/Clone: 3G8), CD56 (Alexa 700/Clone: B159), CD62L (CF594/Clone: Dreg 56), CD38 (FITC/Clone: HIT2), and PD-1 (PerCP-Cy5.5/Clone: EH12.1) from BD Pharmingen (San Jose, CA, USA) and NKG2A (PE/Clone: 131411; R&D, Minneapolis, MN, USA) and Tim-3 (PE-Cy7/Clone: F38-2E2; Biolegend, San Diego, CA, USA). Approximately 70 µL of whole blood was stained for 20 min and then incubated for 15 min with FACS lysing solution (BD FACS Lysing; BD Biosciences, San Jose, CA) to lyse the erythrocytes. After two washes in an isotonic solution (Hemoton SPEC; Brazil), 500,000 events were acquired using a flow cytometer (LSR Fortessa; BD Biosciences, USA) and were analyzed using FlowJo Software (Tree Star, Ashland, OR, USA).
7
Lymphocyte Subset Analysis in Thymoma
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Fasting venous blood (1-2ml) was collected from thymoma patients before and after operation. 50μl heparin and 20μl of six color fluorescent monoclonal antibodies were added to the blood and mixed at room temperature in the dark for 15 minutes. 450μl hemolysin was added to each tube and BD FACS Lysing was used to dilute it. Samples were mixed at room temperature for 10 minutes and then analyzed by flow cytometry and lymphocyte subsets analysis software. T lymphocyte surface markers were CD3+, CD4+, CD8+, and NK cells markers were CD16+ and CD56+.
8
NK Cell Immunophenotyping by Flow Cytometry
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To analyze activation/inhibition markers in the NK cells, venous blood was collected in EDTA anticoagulant tubes, and staining was performed using the following antibodies: CD3 (BV605/clone SK7), CD19 (Horizon V500/clone HIB19), CD16 (APC-Cy7/clone 3G8), CD56 (Alexa 700/clone B159), NKG2D (PECy7/clone 1D11) NKp46 (APC/clone 9-E2) and CD57 (APC/clone NK-1) from BD Biosciences (San Jose, CA, USA) and NKG2A (PE/clone 131491) and NKG2C (Alexa Fluor 488/clone 134591) from R&D Minneapolis, MN, USA. Approximately 70 μL of whole blood was stained for 20 min and then incubated for 15 min with FACS lysing solution (BD FACS Lysing; BD Biosciences) to lyse the erythrocytes. After two washes in an isotonic solution (Hemoton SPEC; Brazil), 300,000 events were acquired using a flow cytometer (LSR Fortessa; BD Biosciences, USA) and were analyzed using FlowJo Software (Tree Star, Ashland, OR, USA).
9
Immunophenotyping Leukocytes from Blood, Spleen, and Liver
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Flow cytometry technique was employed for immunophenotyping leukocytes isolated from blood, spleen and liver samples. The organs were dissociated in wire mesh screens using RPMI medium. Spleen macerates and blood samples were treated with BD FACS Lysing for red blood cell lysis and fixation according to manufacturer's instructions (BD Biosciences, USA). Liver-infiltrated leukocytes were isolated by Ficoll gradient. In this case, liver macerates were gently transferred to tubes containing 5 mL of Ficoll. Samples were spinned down at 800 g for 30 min at room temperature. The mononuclear cell ring and interphase were isolated, washed two times and suspended in PBS pH 7.4. Samples were then treated with BD FACS Lysing solution. Isolated cells were finally washed and suspended in PBS/BSA 1%. Approximately 106 cells were stained on ice for 20 min in the dark with the following mAb combination: CD3-PE, CD4-Alexa Fluor 647, CD8-PerCP and CD45RB-FITC. All mAbs used for this work were obtained from BD Biosciences and background-staining controls were performed using isotypes recommended by the manufacturer. Samples were read in a BD FACS Canto II and analyzed offline with FlowJo (ThreeStar Inc, USA) software.
10
T Cell Maturation Profiling in Peripheral Blood
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To analyze the exhaustion/activation markers at different stages of T cell maturation (naïve CCR7+ CD45RA+, effector CCR7− CD45RA−, memory CCR7+ CD45RA−, memory RA CCR7− CD45RA+) in the peripheral blood, venous blood was collected in EDTA-anticoagulated tubes, and staining was performed using the following antibodies: CD3 BV605 (SK7), CD4 V500 (RPA-T4), CD8 PerCP-Cy5.5 (RPA-T8), CD45RA APC-H7 (HI100), CD127 PE-Cy7 (HIL-7R-M21), CCR7 APC (3D12) and PD-1 PE (MIH4). To analyze follicular helper T cells, CD3 BV605 (SK7), CD4 V500 (RPA-T4), CXCR5 PerCP-Cy5.5 (RF8B2) and PD-1 FITC (MIH4) were evaluated. Antibodies were purchased from BD Pharmingen (San Jose, CA, USA). Approximately 70 µL of whole blood was stained for 20 min and then incubated for 15 min with FACS lysing solution (BD FACS Lysing; BD Biosciences, San Jose, CA) to lyse the erythrocytes. After two washes in an isotonic solution (Hemoton SPEC; Brazil), 300,000 events were acquired using a flow cytometer (LSR Fortessa; BD Biosciences) and were analyzed using FlowJo Software (Tree Star, Ashland, OR, USA).Regulatory T cells were evaluated according to the instructions for the FoxP3 staining kit (eBiosciences, San Diego, CA, USA).
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