Fasting venous blood (5 mL) was collected before and six months preoperatively and centrifuged (3000 r/min, 10 minutes). Serum was separated and cryopreserved (-800C). Immunoturbidimetric method was utilized to detect the levels of immunoglobulin (IgA, IgG, IgM), and the kits used were provided by Changchun Huili Biotechnology Co., LTD. CD3+, CD4+, CD8+ levels were detected by DxFLEX flow cytometry from Beckman Instruments, Inc., USA, and CD4+/CD8+ values were calculated.
Dxflex flow cytometry
Manufactured by Beckman Coulter
Sourced in United States
The DxFLEX flow cytometry system is an automated instrument designed for analyzing and sorting cells and other particles. It utilizes laser technology and sophisticated detectors to measure multiple physical and fluorescent characteristics of individual cells or particles passing through a fluid stream. The DxFLEX is capable of high-speed data acquisition and analysis, providing researchers and clinicians with detailed information about the composition and properties of complex samples.
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Lab products found in correlation
Golgiplug protein transport inhibitor, by BD (1 mentions)
Anti human cd107a antibody, by BD (1 mentions)
Kaluza analysis 2, by Beckman Coulter (1 mentions)
Affinipure f ab 2 fragment specific goat anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions)
Streptavidin pe, by BD (1 mentions)
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysate, by BD (1 mentions)
Anti cd19 fitc, by BD (1 mentions)
Anti cd8 pe antibody, by BD (1 mentions)
Anti cd45 pc7, by BD (1 mentions)
Prism 5, by GraphPad (1 mentions)
Modfitlt v3, by Verity Software House (1 mentions)
Cell cycle kit, by Keygen Biotech (1 mentions)
10 protocols using dxflex flow cytometry
1
Preoperative Immune Biomarker Evaluation
2
Comprehensive Immunological and Nutritional Assessment
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Lymphocyte subpopulation analysis: Flow cytometry method, contained T lymphocytes (CD3+), B lymphocytes (CD3- CD19+), and NK lymphocytes (natural killer cells, CD3- CD16+ and/or CD56+); T cells were subdivided into helper T cells (CD3+ CD4+) and suppressor T cells (CD3+ CD8+). The DxFLEX flow cytometry (lymphocyte division) analyzer (Beckman Coulter Ltd., United States) and lymphocyte assay kit (Beckman Coulter Ltd., United States) were used.
Immunoglobulin + complement, nutritional panel (scattering and turbidimetric method): The nutritional panel contained albumin (ALB), prealbumin (PAB), and transferrin; immunoglobulin and the complement tests contained immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), C3, and C4. BN II analyzer (SIEMENS GmbH, Germany). The IgM assay kit as well as the nutritional complete assay kit (SIEMENS Healthcare Diagnostics GmbH, Germany) were used.
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3
Apoptosis and Cell Cycle Analysis of HUVEC and LLC Cells
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Apoptosis was analyzed with an YF647A-AnnexinV and propidium iodide (PI) apoptosis kit according to the manufacturer’s instruction. Briefly, the HUVECs and LLC cells were inoculated into six-well plates at a density of 5.0 × 104 cells/well. The cells were treated with NS, HA–Tyr, AL, or AL–HA–Tyr for 24 h. After digestion with trypsin and centrifugation for 5 min at 300×g, the cells were washed with cold PBS and centrifuged again. Complete medium was added and incubated at 37 °C for 30 min, and the cells were washed twice and centrifuged for 5 min at 300×g. The cells (1.0 × 105) were collected and re-selected with 100 μL of 1× buffer. Next, 5 μL PI and 5 μL YF647A-Annexin were added to and incubated for 15 min in the dark. The cells were analyzed by DxFlex flow cytometry (Beckman Coulter, Brea, CA). For cell cycle analysis, the treated cells were washed with cold PBS and centrifuged for 5 min at 1000×g. The cells were suspended in 1 mL 75% cold ethanol and fixed overnight at −20 °C. After centrifugation, the cells were washed, centrifuged, and then incubated in the dark with PI and RNaseA staining buffers for 30 min. The stained cells were analyzed by DxFlex flow cytometry.
4
Characterization of Gene-edited CAR T Cells
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Gene‐edited Jurkat CAR19 and CAR T‐19 cells were incubated with a biotin‐SP‐AffiniPure F(ab)’2 fragment‐specific goat anti‐mouse IgG antibody (Jackson ImmunoResearch) to assess CAR expression, followed by incubation with streptavidin‐PE (BD Biosciences). In addition, the gene‐edited Jurkat CAR19 and CAR T‐19 cells were incubated with anti‐human B2M, CD3 (BD Biosciences), HLA‐ABC, HLA‐C, HLA‐E, and HLA‐G (Biolegend) antibodies.
Gene‐edited CAR T‐19 cells were washed and stained with anti‐human CD3, CD4, CD8, CD45RO, and CD62L antibodies (BD Biosciences).
Gene‐edited CAR T‐19 cells were cocultured with Raji or K562 cells at an effector‐to‐target (E:T) ratio of 1:1 together with an anti‐human CD107a antibody (BD Biosciences) for 1 h to assess degranulation, followed by incubation with a Golgi Plug protein transport inhibitor (BD Biosciences) for 3 h.
Primary NK cells were incubated with anti‐human CD3, CD56 (BD Biosciences), NKG2A, KIR2DL4, and LILRB1 (Biolegend) antibodies.
All of the antibody information is presented in Supporting Information Table1 . All samples were assessed using DxFLEX flow cytometry (Beckman Coulter). The data were analyzed with Kaluza Analysis 2.0 (Beckman Coulter) and FlowJo software version 10 (FlowJo LLC).
Gene‐edited CAR T‐19 cells were washed and stained with anti‐human CD3, CD4, CD8, CD45RO, and CD62L antibodies (BD Biosciences).
Gene‐edited CAR T‐19 cells were cocultured with Raji or K562 cells at an effector‐to‐target (E:T) ratio of 1:1 together with an anti‐human CD107a antibody (BD Biosciences) for 1 h to assess degranulation, followed by incubation with a Golgi Plug protein transport inhibitor (BD Biosciences) for 3 h.
Primary NK cells were incubated with anti‐human CD3, CD56 (BD Biosciences), NKG2A, KIR2DL4, and LILRB1 (Biolegend) antibodies.
All of the antibody information is presented in Supporting Information Table
5
Multiplex Cytokine Quantification in Plasma
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The levels of 12 cytokines (IFN-α, IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, and IL-17) in the plasma of patients were measured using cytokine detection kits (Qingdao Raisecare Biological Technology, Qingdao, China). First, 2 mL peripheral blood collected by EDTA-K2 anticoagulant tube was centrifuged at 4°C for 10 min at 1000×g, and plasma was absorbed for later use. Then, 25 μL of experimental buffer, 25 μL of cytokine capture antibody-coated fluorescent microspheres, 25 μL of plasma specimens, and 25 μL of cytokine-detecting antibodies were added to the flow tubes and incubated at 37°C for 2 h. Besides, 25 μL of SA-PE was added and incubated for 0.5 h with shaking at room temperature. After washing with 500 μL of washing buffer, 200 μL of washing buffer was added to the reaction tubes. The expression levels of 12 cytokines were identified by DxFLEX flow cytometry (Beckman Coulter).
6
Apoptosis Measurement in Oxidatively Stressed Caco-2 Cells
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To measure apoptosis in oxidatively stressed cells treated with the fermented beverages, Caco-2 cells were grown in 96-well plates with EMEM medium and different amounts of either fermented beverage (0.25 mg/mL, 1 mg/mL, 2 mg/mL) at 37 °C for 24 h. Cells were treated or not with H2O2 (1 mM) for 2 h at 37 °C, then washed with pre-cooled PBS at 4 °C, and centrifuged at 110× g for 5 min at 4 °C. Supernatants were discarded and 5 μL of Annexin V (Invitrogen, Carlsbad, CA, USA) and 1 μL of 100 μg/mL PI (Invitrogen, Carlsbad, CA, USA) working stocks were added to each cell suspension, then incubated for 15 min at room temperature. Apoptosis was determined using DxFLEX flow cytometry (Beckman-Coulter, Bria, CA, USA).
7
Single-Cell Isolation and Preparation
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Single-cell suspensions were obtained from human ovarian tissue or peripheral blood and from mouse BM, tumor tissue or peripheral blood obtained as described above. Then, the suspensions were centrifuged at 2000 rpm for 10 min. The cell pellets were blocked on ice with PBS buffer containing 2% FBS for 15 min. After centrifugation at 2000 rpm for 10 min, the cells were suspended in a buffer containing primary antibodies and incubated on ice for 30 min in the dark. After removal of any unbound antibodies with a 5 ml volume of complete DMEM, the cell pellets were collected by centrifugation at 2000 rpm for 10 min. Adding 5 mL of red blood cell lysis buffer (Haoyang Biological Manufacture) to the cell pellet and leave it for 10 min at room temperature in the dark, and then the cells were centrifuged at 2000 rpm for 10 min. After the cells were washed with PBS, they were counted and resuspended in a 100 µL flow tube with a filter for FCM (DxFLEX flow cytometry, Beckman Coulter, Brea, California, USA) analysis or fluorescence-activated cell sorting (FACS) (Aria SORP flow sorter, BD Biosciences, Franklin Lakes, New Jersey, USA) sorting.
8
Immunophenotyping of Peripheral Blood
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Peripheral blood samples were surface-labeled with anti-CD19 FITC (BD, California, USA), anti-CD3 PC5.5 (BD, California, USA), anti-CD8-PE antibody (BD, California, USA), or anti-CD45 PC7 (BD, California, USA) for 10 min at room temperature. The red blood cells in the blood were lysed with red blood cell lysate (BD, California, USA), then washed with PBS twice, and detected on the Dxflex Flow cytometry (Beckman, California USA). The results were analyzed using the Kaluza v2.1.1 software.
9
Cell Cycle Analysis of Compound 11r
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Cells were seeded in 96-well plates at the density of 5 × 103 per well and incubated for 12 h. The gradient concentrations of compound 11r or DMSO were added to the plates and incubated for 48 h at 37 °C. The cells were then harvested and washed with pre-cooled PBS, and then centrifuged at 10,000 r/min for 5 min at 4 °C. Cells were fixed with 500 μL 70% ethanol (v/v, 30% PBS) at 4 °C overnight and collected at 800 r/min for 15 min. Cells were washed with PBS and re-suspended in 0.4 mL PBS, and then transferred to a tube at a density of 106/mL. 3 μL of RNase-A was added to the tube with the final concentration of about 50 μg/mL and digested at 37 °C for 30 min. 50 µL of PI was added to the tube with the final concentration of about 65 µg/mL, and incubated on ice bath in the dark for 30 min. The cell cycle distribution was analyzed by DxFLEX flow cytometry (Beckman Coulter, Brea, CA, USA) within 1 h. The data were analyzed using Modfit LT 5.0 software and quantified by GraphPad Prism 5 software. One-way ANOVA and Tukey’s test were used for analysis of the statistical differences between test groups. P values < 0.05 were considered to be significantly different.
10
Cell Cycle Analysis of HCC Cells
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Hepatocellular carcinoma cells were incubated for 48 h after EA (14 and 28 µM) at 37°C and DMSO treatment. The treated cells at 70-80% confluence were fixed with 75% ethanol at 4°C and then incubated in 500 µl PBS containing PI and RNase A (9:1) at 37°C for 60 min (Cell Cycle Kit; Nanjing KeyGen BioTech Co., Ltd.). The cell samples were analyzed using DxFLEX flow cytometry (Beckman Coulter, Inc.). The cell percentage in each cycle was finally evaluated using Modfit-LT V 3.0 software (Verity Software House).
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