NHEK-Ad cells (104) were plated on 6-well cell culture plates (SPL). At 80% confluence, the cells were irradiated with 10 Gy of γ-radiation using a Gammacell® 40 Exactor (Best Theratronics; Ottawa, Canada) at the Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute. Cells were trypsinized, washed, and stained with Dead Cell Apoptosis Kit with Annexin V-Alexa Fluor 488 and PI (BD Bioscience; San Jose, CA, USA) and incubated for 15 min at 37 °C in the dark. Cells were analyzed by MACSQuant flow cytometry (Miltenyi Biotech; Bergisch Gladbach, Germany). TUNEL assay was also used to determine the effect of DeinoPol on radiation-induced cell apoptosis according to the manufacturer’s instructions (Promega). In brief, cells were treated with cold 4% paraformaldehyde for 30 min and 0.3% Triton X-100 was added for 5 min. The TUNEL detecting solution (terminal deoxynucleotidyl transferase and fluorescence solution) was added and allowed to stand for 60 min at 37 °C. Images were acquired using an Olympus IX3 inverted fluorescence microscope (Tokyo, Japan).
Macsquant flow cytometry
Manufactured by Miltenyi Biotec
Sourced in Germany
The MACSQuant flow cytometry system is a compact and automated flow cytometer designed for high-performance cell analysis and sorting. It features advanced optics, multiple lasers, and detection channels to enable comprehensive multiparameter analysis of various cell types and populations.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (2 mentions)
Cd107a pe cy5, by Miltenyi Biotec (2 mentions)
Cd3 pe, by Miltenyi Biotec (2 mentions)
Cd25 apc, by Miltenyi Biotec (2 mentions)
Cd56 fitc, by Miltenyi Biotec (2 mentions)
Cd8 vioblue, by Miltenyi Biotec (2 mentions)
Cd4 viogreen, by Miltenyi Biotec (2 mentions)
Cd19 pe cy7, by Miltenyi Biotec (2 mentions)
Gammacell 40 exactor, by Best Theratronics (1 mentions)
Tunel assay, by Promega (1 mentions)
Dead cell apoptosis kit, by BD (1 mentions)
Ix3 inverted fluorescence microscope, by Olympus (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rnase a, by Merck Group (1 mentions)
Hemavet 950, by Drew Scientific (1 mentions)
Fixation and intracellular staining permeabilization wash buffer, by BioLegend (1 mentions)
Human ifn γ elisa kit, by Thermo Fisher Scientific (1 mentions)
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
7 aad, by Thermo Fisher Scientific (1 mentions)
Microplate reader, by BMG Labtech (1 mentions)
8 protocols using macsquant flow cytometry
1
Evaluation of Radiation-Induced Apoptosis
2
Apoptosis Induction in BMCs and Spleen Cells
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BMCs and spleen cells (3×106) were plated on 12-well culture plates with or without 50 μg/ml DeinoPol-BRD125 for 2 h followed by 2 Gy X-ray irradiation. Apoptotic cells were counted by staining with propidium iodide (PI) (Sigma-Aldrich). Briefly, cultured cells were washed once with PBS and fixed with 70% ethanol at -20°C overnight. The fixed cells were resuspended in 1 ml of PI buffer [0.5 mg/ml RNaseA (Sigma-Aldrich) and 0.1 mg/ml PI (Sigma-Aldrich) in PBS]. After incubation for 30 minutes in the dark, the PI of individual nuclei were analyzed by MACSQuant flow cytometry (Miltenyi Biotech; Bergisch Gladbach, Germany).
3
Hematopoiesis Rescue by DeinoPol-BRD125
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The effects of DeinoPol-BRD125 on hematopoiesis were studied in vivo using BM cells transplantation assay. Donor mice were intraperitoneally injected with PBS or DeinoPol-BRD125 at 36 h and 12 h before irradiation with 4 Gy, and 30 min, 24 h, and 48 h after irradiation. On day 7 after irradiation, BM cells from donor mice were harvested and 2×106 BM cells were injected into the tail veins of recipient mice, which were lethally irradiated (8 Gy) 6 h earlier. Two and four weeks after transplantation, cells in peripheral blood were counted using an automatic blood analyzer (Hemavet 950; Drew Scientific; Miami Lakes, Florida, USA), and cell populations in the spleen were analyzed by MACSQuant flow cytometry (Miltenyi Biotech).
4
Multiparametric Flow Cytometry of T Cells
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PE‐labeled anti‐CCR7 (4B12; BioLegend), FITC‐labeled anti‐CD4 (GK1.5; TONBO Bioscience, San Diego, CA, USA), and PE‐Cy5‐labeled anti‐CD8a (53–6.7; TONBO Biosciences) antibodies were used to stain cell‐surface molecules after blocking the Fc receptors with 2.4G2 (BD Pharmingen, Franklin Lakes, NJ, USA). In the experiment described in Fig. 2C , CD4+ T cells were fixed and permeabilized with Fixation and Intracellular Staining Permeabilization Wash Buffer (BioLegend). Fluorescence intensity was acquired by MACSQuant flow cytometry (Miltenyi Biotec, Tubingen, Germany) and analyzed by FlowJo (TOMY Digital Biology, Tokyo, Japan).
5
Preclinical Evaluation of CAR-T Cell Efficacy
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Preclinical in vitro studies with CAR-T cells were performed for the assessment of efficacy and cytotoxic capacity. For that purpose, anti-CD19-expressing CAR-T cells and CD19-expressing RAJI cells (Burkitt’s lymphoma, CCL-86, ATCC®) were co-cultured for 24 h, 7 days, and 14 days (effector:target; 1:1, 5:1, 10:1) prior to labeling target cells including RAJI, DAUDI (ATCC® CCL-213™), and K562 (ATCC® CCL-243) cell lines with either the CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions or α-CD19-PE (BioLegend) and α-CD19-PE.cy7 (Miltenyi) for the analysis with flow cytometry. At the end of the co-culture process, α-EGFR-FITC (Abcam) and 7-AAD (Invitrogen) were evaluated for cytotoxicity. CAR-T cell activation was determined with the upregulation of CD25 (IL2RA, IL-2 receptor alpha chain) and CD107a (marker for degranulation of lymphocytes) on CAR-T cells using α-CD3-PE, α-CD4-Viogreen, α-CD8-Vioblue, α-CD25-APC, and α-CD107a-PE.cy5.5 (Miltenyi) with MACSQuant flow cytometry (Miltenyi). After 24 h of co-culturing, supernatants from cultured cell conditions were also collected and IFNγ cytokine levels were assessed with the Human IFNγ ELISA Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The ELISA plate was measured at 450 nm and 550 nm using a microplate reader (BMG LABTECH).
6
Antibody Binding Evaluation in E. coli
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The binding capability of the four antipeptides antibodies was tested over a pks+ (E. coli Nissle 1977) and a pks− (E. coli LMG2092) strains.
About 25 µL of the bacterial suspension was mixed with 25 µL of the FITC-conjugated antibody at a final concentration of 20 µg/mL. The samples were incubated for 15 min at RT and were then washed with FC buffer at 13,000 rpm for 5 min. Finally, bacteria were resuspended in 150 µL of FC buffer and samples were acquired using a MACSQuant Flow Cytometry (Miltenyi Biotec, Germany). Bacteria were labeled and analyzed at a different optical density (i.e., OD600=0.3, 0.6, 1, and 1.5) and with a different medium (i.e., LB, Nutrient Broth, and Nutrient Broth supplemented with 2% of glucose).
About 25 µL of the bacterial suspension was mixed with 25 µL of the FITC-conjugated antibody at a final concentration of 20 µg/mL. The samples were incubated for 15 min at RT and were then washed with FC buffer at 13,000 rpm for 5 min. Finally, bacteria were resuspended in 150 µL of FC buffer and samples were acquired using a MACSQuant Flow Cytometry (Miltenyi Biotec, Germany). Bacteria were labeled and analyzed at a different optical density (i.e., OD600=0.3, 0.6, 1, and 1.5) and with a different medium (i.e., LB, Nutrient Broth, and Nutrient Broth supplemented with 2% of glucose).
7
Dornase alfa and MgSO4 Effects on PBMC
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Three healthy adult blood was obtained from Acibadem Labcell Cellular Therapy Laboratory.
Following the isolation of peripheral mononuclear cells (PBMC) by overlaying blood on Ficollpaque plus (GE Healthcare), the serially diluted (300 U/mL, 100 U/mL, 30 U/mL, 10 U/mL, 3 U/mL) Dornase alfa (Pulmozyme®, Roche, USA) and 420 g/mL MgSO 4 (Magnesium Sulphate 15% Onfarma, TR) were incubated with the PBMCs for 48hr in T cell medium (6% Human AB Serum and 1% Pen/Strep, Texmacs medium). Immune cell subtypes and their activation levels were determined by Miltenyi MACSQuant flow cytometry analysis using CD3-PE, CD19-PE.cy7, CD56-FITC, CD4-Viogreen, CD8-Vioblue, CD25-APC and CD107a-PE.cy5.5 (Miltenyi).
Following the isolation of peripheral mononuclear cells (PBMC) by overlaying blood on Ficollpaque plus (GE Healthcare), the serially diluted (300 U/mL, 100 U/mL, 30 U/mL, 10 U/mL, 3 U/mL) Dornase alfa (Pulmozyme®, Roche, USA) and 420 g/mL MgSO 4 (Magnesium Sulphate 15% Onfarma, TR) were incubated with the PBMCs for 48hr in T cell medium (6% Human AB Serum and 1% Pen/Strep, Texmacs medium). Immune cell subtypes and their activation levels were determined by Miltenyi MACSQuant flow cytometry analysis using CD3-PE, CD19-PE.cy7, CD56-FITC, CD4-Viogreen, CD8-Vioblue, CD25-APC and CD107a-PE.cy5.5 (Miltenyi).
8
Dornase alfa and MgSO4 Effects on PBMC
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Three healthy adult blood was obtained from Acibadem Labcell Cellular Therapy Laboratory.
Following the isolation of peripheral mononuclear cells (PBMC) by overlaying blood on Ficollpaque plus (GE Healthcare), the serially diluted (300 U/mL, 100 U/mL, 30 U/mL, 10 U/mL, 3 U/mL) Dornase alfa (Pulmozyme®, Roche, USA) and 420 g/mL MgSO 4 (Magnesium Sulphate 15% Onfarma, TR) were incubated with the PBMCs for 48hr in T cell medium (6% Human AB Serum and 1% Pen/Strep, Texmacs medium). Immune cell subtypes and their activation levels were determined by Miltenyi MACSQuant flow cytometry analysis using CD3-PE, CD19-PE.cy7, CD56-FITC, CD4-Viogreen, CD8-Vioblue, CD25-APC and CD107a-PE.cy5.5 (Miltenyi).
Following the isolation of peripheral mononuclear cells (PBMC) by overlaying blood on Ficollpaque plus (GE Healthcare), the serially diluted (300 U/mL, 100 U/mL, 30 U/mL, 10 U/mL, 3 U/mL) Dornase alfa (Pulmozyme®, Roche, USA) and 420 g/mL MgSO 4 (Magnesium Sulphate 15% Onfarma, TR) were incubated with the PBMCs for 48hr in T cell medium (6% Human AB Serum and 1% Pen/Strep, Texmacs medium). Immune cell subtypes and their activation levels were determined by Miltenyi MACSQuant flow cytometry analysis using CD3-PE, CD19-PE.cy7, CD56-FITC, CD4-Viogreen, CD8-Vioblue, CD25-APC and CD107a-PE.cy5.5 (Miltenyi).
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