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8 protocols using fcs express

1

CD34+ Cells Enumeration in Blood

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Within 24 h of blood collection, 100 μL of blood from each donor was used to count CD34+ cells by BD Procount progenitor cell enumeration kit (BD Biosciences, San Jose, CA) per manufacturer instructions followed by analysis on a LSR II flow cytometer (BD Biosciences) with FCS Express (BD Biosciences).
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2

CD44 and CD24 Expression Analysis

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1X 106 cells were labeled with PE-conjugated anti-CD44 (Biolegend, San Diego, USA) and FITC-conjugated CD24 (BD Pharmingen, Mississauga, Canada) for 20 min, washed twice, resuspended in PBS and analyzed on a BD FACSCalibur™ platform (Beckon Dickinson, Quebec, Canada) or MACS Quant Analyzer (Miltenyi Biotec, Cologne, Germany). Data was analyzed by FCS Express (BD Bioscience) or MACS Quantify (Miltenyi Biotec) softwares using floating quadrants to enumerate negative, single-positive and double-positive populations. CD44 and CD24 double-labeling studies always included double negative and single positive staining controls for compensation. Immunocytochemistry was performed as described before. Staining was visualized by Zeiss LSM700 confocal laser scanning microscopy system or Nikon inverted microscope.
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3

Cellular Uptake of Verteporfin

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Cells were plated on 6-well plates and treated with verteporfin or NLC-verteporfin for 2 h or 24 h at 37 °C or at 4 °C in the dark. After incubation cells were harvested and re-suspended in 1× PBS for flow cytometry analysis. Fluorescence emission of verteporfin was analyzed using flow cytometry LSRII and FCS Express software (BD Biosciences, San Jose, CA, USA).
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4

3D Bioreactor Levitation Culture Model

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For the establishment of a three dimensional model, the bench-top bioreactor BioLevitator (Hamilton Company, Switzerland) and basal membrane coated GEM were used. Cells were incubated in specialized culture vessels (LeviTubes; Hamilton)) in the bioreactor at 37°C and 5% CO2. 3×106 cells were seeded on 1 ml GEM pre-coated with collagen IV or gelatine (Omni Life Science, Bremen, Germany) in a total volume of 10 ml medium with 10% FBS. After an overnight inoculation period, LeviTubes were filled with an additional 15 ml of medium. An aliquot of the microcarrier cultures was treated with ultrasonic irradiation, 1 h later GEM were dissolved in pre-warmed trypsin/EDTA, GEM were removed by a magnet, the restraining cells were stained with PI measurement and analysed by FACSCalibur (BD Biosciences, San Jose, US) and by FCSexpress De Novo (Glendale, CA) as described above. Staining was additionally documented on a LSM510 Meta confocal laser scanning microscope (Zeiss, Germany).
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5

Assessing Small Molecule Toxicity in mESCs

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To assess the toxicity of the small molecules, mESC cells with stably integrated gRNAs from the 48-site library were transfected with p2T CAG Cas9 BlastR and KU-0060648, KU-60019, or DMSO as indicated. After 24 h of transfection, cells were trypsinized, stained with 0.5 µg/mL propidium iodide (PI, Biolegend), and subjected to flow cytometry using a Cytek DXP11 using a consistent flow-rate. Data were analyzed using BD FACSDiva and FCS Express software, and the PI gating parameters were obtained using a live-cell gate set using untreated cells and 0.25% Triton X-100-treated cells (Supplementary 4b).
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6

Phosphoflow Analysis of PBMC Signaling

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PBMCs from healthy patients were treated with each drug for 2 hours and stimulated with 3.3 mM H2O2 for 10 minutes at 37°C. The cells were then fixed with 1.6% paraformaldehyde solution for 10 minutes at 37°C and permeabilized with 100% methanol overnight at –80°C. Cells were stained with antibodies against phospho-LCK (#558552; BD Biosciences, Franklin Lakes, NJ), phospho-SRC (#560096; BD Biosciences), cleaved Poly(ADP-ribose) polymerase (PARP) (#560640; BD Biosciences) and CD3 (#562426; BD Biosciences). A similar assay was previously described for T-cell phosphoflow analysis using CD3/CD28 and H2O2 stimuli (38 (link),39 (link)). Data were acquired on a FACSVerse flow cytometer and analyzed with FCS Express (version 4; BD Biosciences).
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7

Comprehensive Preclinical and Clinical Data Analysis

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GraphPad Prism 8 was used for all pre-clinical data analysis, utilizing two-tailed statistical tests with the assumption of normality. The application of two-way analysis of variance with Bonferroni correction was used to account for time and the different CAR cohorts. Flow cytometry data were analyzed using FlowJo v10.1.1, FCS Express v7.06.0015 and BD FACSuite v1.3. Transcriptomics data were analyzed using nSolver v2.0.134 and R studio 3.6. Clinical data are captured in the clinical database via the Encapsia electronic data capture (EDC) system v1.0. SAS 9.4 was used for clinical data analysis. Categorical variables are reported in terms of frequency and percentage, and continuous variables in terms of median and range unless otherwise specified. Time-to-event outcomes were summarized using the Kaplan–Meier method. Toxicity events are reported at the maximum grade experienced according to the CTCAE. The cellular kinetics parameters were estimated from the individual concentration versus time profiles using a non-compartmental approach in Phoenix WinNonlin.
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8

Comprehensive Bone Marrow Flow Cytometry

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Flow cytometry was performed on aspirated bone marrow collected in Roswell Park Memorial Institute (RPMI) culture medium. Cells were processed routinely, washed in Dulbecco’s phosphate-buffered saline (DPBS), labeled with antibodies, treated with ammonium chloride RBC lysis buffer (Pharmlyse; BF Biosciences, San Jose, CA), and then washed again with DPBS. In the case of cytoplasmic or nuclear staining antibodies, samples were also permeabilized during antibody labeling. Antibodies against CD19 and cytoplasmic and surface CD3 were used in all cases. Most cases also were stained with antibodies against B cell antigens CD79a, CD22, CD10, and CD20, T cell antigens CD2, CD4, CD5, CD7, CD8, and CD56, and the myelomonocytic markers MPO, CD11b, CD11c, CD33, CD13, CD14, CD15, and CD64. Samples were run on CANTO II or CALIBUR Flow Cytometers (BD Biosciences) and analyzed using FACSDiva (BD Biosciences), FCS Express (BD Biosciences), and/or FlowJo (FlowJo, Ashland, OR).
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