Flow cytometry of absolute counts for T cells were analyzed in Trucount tubes (BD, Franklin Lakes, NJ, USA) on a FacsCanto II instrument and analyzed in BD FACSCanto™ Clinical Software according to the instructions provided by the manufacturer (BD). Instrument settings were standardized as recommended by the manufacturer with daily quality run with CS&T Beads (BD) and 7-color Setup Beads (BD) ensuring high reproducibility. The laboratory follows standard operation procedure and also has ISO (International Standard Organization) certification. Further sub-classification of T cells was performed on a Gallios Flow cytometer (Beckman Coulter, San Diego, CA, USA) as previously described (9 (link)). Reference values (5–95 percentile) for absolute numbers of T and NK-cells, and T-subpopulations were established on samples from healthy blood donors (n = 65).
7 color setup beads
Manufactured by BD
The 7-color Setup Beads are a set of particles specifically designed to calibrate and set up flow cytometry instruments. These beads are used to verify the instrument's performance and ensure accurate measurements across seven distinct fluorescence channels.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (2 mentions)
Cs t beads, by BD (1 mentions)
Trucount tube, by BD (1 mentions)
Facscanto clinical software, by BD (1 mentions)
Gallios flow cytometer, by Beckman Coulter (1 mentions)
Facsflow, by BD (1 mentions)
Cd4 pe cy7, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Diva software, by BD (1 mentions)
Cd86 pe it2, by BD (1 mentions)
Hla dr percp l243, by BD (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
Cd38 apc hb 7, by BD (1 mentions)
Cd3 amcyam clone sk7, by BD (1 mentions)
Facscanto, by BD (1 mentions)
3 protocols using 7 color setup beads
1
Absolute T Cell Counts by Flow Cytometry
2
Multicolor Flow Cytometry of Immune Cell Activation
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EDTA anticoagulated peripheral blood cells were labeled following a lyse/wash protocol with an 8-color/9-monoclonal antibody (mAb) panel including CD3 AmCyam (clone SK7, BD Biosciences), CD4 PECy7 (SK3, BD), CD8 APCCy7 (SK1, BD), CD16 PacBlue (3G8, BD), CD19 PECy7 (SJ25C1, BD), CD28 FICT (CD28.2, BD), CD38 APC (HB7, BD), CD86 PE (IT2.2, BD), and HLA-DR PerCP (L243, BD). Five microliters of each antibody in 100 µL of whole blood was incubated for 15 minutes at room temperature in the dark. Samples were lysed with 3 mL of 1X FACSlysing solution (BD) for 5 minutes and washed with 3 mL of FACSFlow (BD). Half a million cells were immediately acquired in a FACSCanto flow cytometer (BD), daily calibrated using 7-color setup beads (BD), and analyzed with DIVA software (BD) following the gating strategy described in Supplementary Figure 1 .
The expression of CD28, CD38, CD86, and HLA-DR activation/senescence markers was evaluated as a percentage or absolute number (cells/µL) of positive cells as well as mean fluorescence intensity (MFI) of the marker on CD3+CD4+, CD3+CD8+, and CD3+CD4+CD8+ T lymphocytes, CD19+ B lymphocytes, CD3-CD19-CD16+ natural killer (NK) lymphocytes, monocytes (CD4+CD86+HLA-DR+ medium side scatter [SSC] cells), granulocytes (CD16++ elevated SSC cells), and eosinophils (elevated SSC auto fluorescent cells).
The expression of CD28, CD38, CD86, and HLA-DR activation/senescence markers was evaluated as a percentage or absolute number (cells/µL) of positive cells as well as mean fluorescence intensity (MFI) of the marker on CD3+CD4+, CD3+CD8+, and CD3+CD4+CD8+ T lymphocytes, CD19+ B lymphocytes, CD3-CD19-CD16+ natural killer (NK) lymphocytes, monocytes (CD4+CD86+HLA-DR+ medium side scatter [SSC] cells), granulocytes (CD16++ elevated SSC cells), and eosinophils (elevated SSC auto fluorescent cells).
3
Immunophenotyping of Lymphocyte Subsets
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Based on the expression of surface markers, immunophenotyping allows quantification by flow cytometry of main lymphocyte subsets from whole hemolyzed blood: T lymphocytes (CD45+CD3+), B lymphocytes (CD45+CD3-CD19+), helper T lymphocytes (CD45+CD3+CD4+), suppressor/cytotoxic T lymphocytes (CD45+CD3+CD8+), and NK cells (CD45+CD3-CD16+CD56+).
In order to determine the percentages of these subsets, a BD Multitest IMK Kit (IVD) (Becton Dickinson) was used. EDTA-anticoagulated whole peripheral blood was incubated with a mixture of monoclonal antibodies (CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC; CD3-FITC/CD16+CD56-PE/CD45-PerCP/CD19-APC) for 15 min at room temperature and in the dark, followed by red blood cell lysis and flow cytometry analysis (BD FACSCanto II, Becton Dickinson). BD FACSCanto clinical software was used for sample acquisition and data analysis; daily check-up of cytometer performances was performed using 7-Color Setup Beads (BD Biosciences).
In order to determine the percentages of these subsets, a BD Multitest IMK Kit (IVD) (Becton Dickinson) was used. EDTA-anticoagulated whole peripheral blood was incubated with a mixture of monoclonal antibodies (CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC; CD3-FITC/CD16+CD56-PE/CD45-PerCP/CD19-APC) for 15 min at room temperature and in the dark, followed by red blood cell lysis and flow cytometry analysis (BD FACSCanto II, Becton Dickinson). BD FACSCanto clinical software was used for sample acquisition and data analysis; daily check-up of cytometer performances was performed using 7-Color Setup Beads (BD Biosciences).
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