PBMCs were thawed, washed twice, and stained for 20 min at room temperature for flow cytometric analysis and cell sorting with the following antibodies: LIVE/DEAD Fixable Green Dead cell Stain Kit, Thermofisher or DAPI; CD14-FITC, clone M5E2, BD Biosciences; CD19-FITC, clone HIB19, BD Biosciences; γδ TCR-PE, clone 11F2, eBiosciences; Vγ9-PE-Cy5, clone IMMU 360, Beckman Coulter; Vδ2-APC, clone 123R4, Miltenyi; CD45-APC-Cy7, clone 5B1, Miltenyi; CD3-BV786 and CD3-PECy7, clone UCHT1, BD Biosciences. After staining, PBMCs were washed twice and stored on ice until acquisition and sorting on a FACSAria Fusion cell sorter (BD Biosciences). HC and patients recruited at the Department of Gastroenterology, Hepatology and Endocrinology were sorted for CD14−/CD19− γδ T cells and HC recruited at the Institute for Immunology were sorted for CD45+/CD3+ γδ T cells. Flow cytometry data were analyzed using the Flow Jo software V.9.8 (Tree Star Inc., Ashland, OR, USA).
Cd19 fitc clone hib19
Manufactured by BD
Sourced in United States
CD19 FITC (clone HIB19) is a fluorochrome-conjugated monoclonal antibody used for the detection and identification of CD19-positive cells in flow cytometry analysis. CD19 is a cell surface antigen expressed on B cells and B cell precursors.
Automatically generated - may contain errors
Lab products found in correlation
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (2 mentions)
Facsaria iiiup, by BD (2 mentions)
Live dead fixable green dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Facsaria fusion cell sorter, by BD (1 mentions)
Acuri c6, by BD (1 mentions)
Cd3 fitc clone hit3a, by BD (1 mentions)
Cd19 pe clone hib19, by BD (1 mentions)
Cd4 pe clone rpa t4, by BD (1 mentions)
Cd8 apc clone rpa t8, by BD (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Facscalibur instrument, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cellquest software, by BD (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Facs lysing solution, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Pharm lyse lysing solution, by BD (1 mentions)
6 protocols using cd19 fitc clone hib19
1
Isolation and analysis of human γδ T cells
2
Measuring MET-CAR Transduction Efficacy
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To measure MET-CAR transduction efficacy, cells were collected on day 5 after transduction, washed with PBS containing 1% FBS, and incubated with CD3 and CD19 antibodies for 30 min followed by analysis using a flow cytometer (Acuri C6 + , BD). To determine the subsets of MET-CAR-T cells, CD19+ T cells were gated for CD4+ and CD8+ populations. To determine programmed cell death protein 1 (PD-1) upregulation in MET-CAR-T cells, non-transduced (NT) or MET-CAR-T cells were co-cultured with MHCC97H cells in 24-well plates (105 cells/well) at a 1:1 ratio for 3 days. At day 0 (before co-culture) and day 3, NT (gated by CD3+) and MET-CAR-T cells (gated by CD19+) were harvested to measure the PD-1 expression using flow cytometry analysis. Antibodies and isotype controls used in the analysis include: CD3 FITC (clone HIT3A, BD), CD19 PE (clone HIB19, BD), CD19 FITC (clone HIB19, BD), CD4 PE (clone RPA-T4, BD), CD8 APC (clone RPA-T8, BD), PD-1 Cy7(Clone EH12.1, BD), IgG2b κ FITC (clone 27-35, BD), IgG1 PE (clone MOPC-21, BD), IgG2b κ PE-Cy7 (clone 27-35, BD), and IgG1 APC (clone P3.6.2.8.1, Invitrogen, San Diego, CA).
3
Evaluating Leukemic Cell Viability
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The viability of the leukemic cells was also evaluated with the LIVE/DEAD Fixable Aqua Dead Cell Stain (Molecular Probes, Eugene, OR, USA). After incubation of MSC with the PKC inhibitors, the co-cultures were stablished for the indicated time-periods and then double labelled with CD19 FITC (clone HIB19, BD Pharmingen, San Jose, CA, USA) and the LIVE/DEAD Aqua stain for viability evaluation by flow cytometry (FACSAria IIIup Becton Dickinson Biosciences, San Jose, CA, USA); data were analyzed with the FlowJo software. First, the leukemic cell line RS4;11 was evaluated alone or in the co-culture with MSC pre-treated or not with the PKC inhibitors STAU and ENZA. Then, evaluations were performed in B-ALL cells from patients, as follows: 5 × 104 B-ALL cells were seeded in 96-well microplates containing confluent MSC that have been previously treated with the PKC inhibitors or peptides. MSC viability was also evaluated in the co-cultures by labelling with LIVE/DEAD Aqua and CD105 and non-treated MSC were used as controls. Cell viability was determined either after 2 or 48 h of incubation as described above.
4
Overcoming Glucocorticoid Resistance in B-ALL
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Leukemic cells derived from patients (n = 5) that had shown a worse response at the final of the induction treatment were chosen for these experiments. B-ALL cells were treated during 6 h with 250 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) and then cells were co-cultured on 70% confluent MSC that have been pre-treated or not for 2 h with 40 µM HKPS, HK or 20 µM ENZA. Three days later, the unattached B-ALL cells were collected and the co-cultures were washed and trypsinized, and cells thus recovered were mixed with the corresponding unattached cells and double labelled with CD19 FITC (clone HIB19, BD Pharmingen, San Jose, CA, USA) and the LIVE/DEAD Fixable Aqua Dead Cell Stain (Molecular Probes, Eugene, OR, USA); evaluations and analysis were performed by flow cytometry (FACSAria IIIup Becton Dickinson Biosciences, San Jose, CA, USA) using the FlowJo software.
5
Quantification of Soluble APRIL Levels
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Soluble APRIL plasma levels were measured by a commercial Human APRIL ELISA kit (Invitrogen, Carlsba, CA, USA) We followed the protocols recommended by the manufacturer. The ELISA Reader Victor 3 (PerkinElmer, USA) was used.
Mononuclear cells were separated by density gradient centrifugation with Biocoll Separating Solution (Biochrom, Harvard Bioscience, Holliston, MA 01746, US) for 25 minutes at 400 × g at room temperature. Interphase cells were removed, washed twice, and resuspended in phosphate-buffered saline (PBS).
For intracellular detection of APRIL, PBMC were stained with MAb against cell surface markers, i.e. CD19 FITC (Clone HIB19, BD Pharmingen, San Diego, CA, USA) (20 minutes at room temperature). Following membrane staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution and Perm/Wash buffer (BD Pharmingen, San Diego, CA, USA ) according to the manufacturer’s instructions. Cells were then incubated for 30 mins at RT with PE-conjugated anti-APRIL MAb (Clone: A3D8, BioLegend, San Diego, CA, USA) or with an isotype control antibody. Finally, cells were washed and analyzed by flow cytometry directly after preparation. A FACSCalibur instrument (Becton Dickinson Franklin Lakes, NJ, USA) and CellQuest software (Becton Dickinson Franklin Lakes, NJ, USA) were used.
Mononuclear cells were separated by density gradient centrifugation with Biocoll Separating Solution (Biochrom, Harvard Bioscience, Holliston, MA 01746, US) for 25 minutes at 400 × g at room temperature. Interphase cells were removed, washed twice, and resuspended in phosphate-buffered saline (PBS).
For intracellular detection of APRIL, PBMC were stained with MAb against cell surface markers, i.e. CD19 FITC (Clone HIB19, BD Pharmingen, San Diego, CA, USA) (20 minutes at room temperature). Following membrane staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution and Perm/Wash buffer (BD Pharmingen, San Diego, CA, USA ) according to the manufacturer’s instructions. Cells were then incubated for 30 mins at RT with PE-conjugated anti-APRIL MAb (Clone: A3D8, BioLegend, San Diego, CA, USA) or with an isotype control antibody. Finally, cells were washed and analyzed by flow cytometry directly after preparation. A FACSCalibur instrument (Becton Dickinson Franklin Lakes, NJ, USA) and CellQuest software (Becton Dickinson Franklin Lakes, NJ, USA) were used.
6
Isolation and Characterization of White Blood Cells
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White blood cells (WBC) were obtained by selective lysis of red bloods cells using either BD Pharm Lyse lysing solution (BD Bioscience) for viable, non-fixed WBC samples, or BD FACS lysing solution (BD Bioscience) for fixed WBC preparation according to manufacture instructions. Cells were stained with monoclonal antibodies for 15 min at room temperature in the dark and adjusted to 1 × 10 6 cells per ml in 1× PBS + 2 mM EDTA + 0.01% BSA (Sigma-Aldrich). The following directly conjugated monoclonal antibodies were used: CD3-APC (clone HIT3a), CD14-PE (clone MφP9), CD19 FITC (clone HIB19), CD45-PerCP (clone 2D1), and CD66b FITC (clone G10F5), as well as matched isotype controls (all from BD Bioscience). Propidium iodide (Sigma-Aldrich) was used as a dead cell marker in viable WBC experiments. Immunofluorescent labeled samples were analyzed before and after acoustic separation on a FACSCanto II flow cytometer (BD Bioscience) and the acquired data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).
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