MMP was assessed using the JC-1 mitochondrial membrane potential assay kit (C2006, Beyotime, Shanghai, China) as described in a previous study [42 (link)]. HUVECs were collected after treatment with melatonin in the presence of ox-LDL. The cells were then incubated with 5 μM JC-1 staining kit reagent at 37°C for 30 minutes in the dark [43 (link)]. Finally, cells were analyzed using the Guava easyCyte Benchtop Flow Cytometer (BR168323; Luminex, Austin, TX, USA) with Kaluza C Analysis Software (version 2.1, Beckman Coulter, Indianapolis, IN, USA) [44 (link)].
Kaluza c analysis software
Manufactured by Beckman Coulter
Sourced in United States
Kaluza C Analysis Software is a data analysis tool designed for flow cytometry applications. It provides users with the ability to analyze and interpret flow cytometry data.
Automatically generated - may contain errors
Lab products found in correlation
Guava easycyte benchtop flow cytometer, by DiaSorin (4 mentions)
Navios flow cytometer, by Beckman Coulter (2 mentions)
Jc 1 mitochondrial membrane potential assay kit, by Beyotime (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
Anti mouse cd4 pe, by Thermo Fisher Scientific (1 mentions)
Fitc anti mouse cd3, by BioLegend (1 mentions)
Ab150157, by Abcam (1 mentions)
Ab218745, by Abcam (1 mentions)
Ab200568, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Ab150115, by Abcam (1 mentions)
Annexin 5 fitc pi cell apoptosis kit, by Beyotime (1 mentions)
10 protocols using kaluza c analysis software
1
Mitochondrial Membrane Potential Assay
2
Apoptosis Detection in MCF7 Cells
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The MCF7 cells (1 × 105) were treated with Annexin V and propidium iodide (PI) (5 μl for each) together for 15 min at room temperature in the dark. Next, the cells were collected and washed by cold PBS twice, and cell apoptosis was detected using Annexin V-FITC/PI cell apoptosis kit (KA3805, Abnova, Walnut, CA, U.S.A.). Cell apoptosis was detected by Guava easyCyte Benchtop Flow Cytometer (BR168323; Luminex, Austin, TX, U.S.A.). The data were analyzed using Kaluza C Analysis Software (Beckman Coulter, Indianapolis, IN, U.S.A.).
3
Multi-parameter Flow Cytometry Immunophenotyping
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Immunophenotyping assays were performed by URMC Clinical Flow Cytometry Laboratory for standard clinical care, using a Beckman Coulter Navios Flow Cytometer, FDA approved 10-Color ClearLLab lyophilized immunophenotyping tubes and Kaluza C analysis software (Beckman Coulter Life Sciences). Peripheral blood samples were processed using a stain/lyse/wash protocol. Cell concentrations were adjusted to 3–20 × 106/mL to ensure optimal antibody staining and the cells were washed three times before acquisition. Viability was assessed using 7AAD and CD45. Following morphological review of peripheral slide, 10-color analyses were performed for the following surface and cytoplasmic antigens: Kappa, Lambda, CD10, CD5, CD200, CD34, CD38, CD20, CD19, CD45, TCR alpha/beta, TCR delta/gamma, CD4, CD2, CD56, CD7, CD8, CD3, CD45, CD16, CD7, CD13, CD64, CD34, CD14, HLA-DR, CD11b, CD15, CD123, CD117, CD33, CD45, 6AC1: cy-TdT, cy-79a, CD22, 6AC2: cy-MPO, CD1a, cy-CD3. Cells were gated to exclude debris (forward scatter vs. side scatter and time of flight), to exclude cell doublets (forward scatter height vs. forward scatter width) and to isolate leukocyte populations (CD45 vs. side scatter). Primary analysis and quality control was performed by the flow cytometry supervisor. Final gating and reporting were performed by a board-certified hematopathologist.
4
Immunophenotyping Assay for Hematology Analysis
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Immunophenotyping assays were performed by URMC Clinical Flow Cytometry Laboratory for standard clinical care, using a Beckman Coulter Navios Flow Cytometer, FDA approved 10-Color ClearLLab lyophilized immunophenotyping tubes and Kaluza C analysis software (Beckman Coulter Life Sciences). Peripheral blood samples were processed using a stain/lyse/wash protocol. Cell concentrations were adjusted to 3–20 × 106/mL to ensure optimal antibody staining and the cells were washed three times before acquisition. Viability was assessed using 7AAD and CD45. Following morphological review of peripheral slide, 10-color analyses were performed for the following surface and cytoplasmic antigens: Kappa, Lambda, CD10, CD5, CD200, CD34, CD38, CD20, CD19, CD45, TCR delta/gamma, CD4, CD2, CD56, CD7, CD8, CD3, CD45, CD16, CD7, CD13, CD64, CD34, CD14, HLA-DR, CD11b, CD15, CD123, CD117, CD33, CD45, 6AC1: cy-TdT, cy-79a, CD22, 6AC2: cy-MPO, CD1a, cy-CD3. Cells were gated to exclude debris (forward scatter versus side scatter and time of flight), to exclude cell doublets (forward scatter height versus forward scatter width), and to isolate leukocyte populations (CD45 versus side scatter). Primary analysis and quality control were performed by the flow cytometry supervisor. Final gating and reporting were performed by a board-certified hematopathologist.
5
Flow cytometric analysis of PD-L1/PD-L2 expression
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Cultured cells were trypsinized, washed with FACS buffer (PBS with 1% BSA) three times and stained for 30 min on ice with respective antibodies: human PD-L1 FITC (Cat: 53-5983-42) and PD-L2 APC (Cat: 17-5888-42) were from Thermo Fisher Scientific (Waltham, MA, USA), and mouse PD-L1 PE was purchased from BioLegend (Cat: 124308, San Diego, CA, USA).
Mouse spleens were collected after sacrifice and dissociated by scalpel. A cell strainer (40 µm) was used to remove the debris. Isolated splenocytes were washed with PBS and subjected to RBC lysis using RBC lysis buffer (Thermo Fisher Scientific, Cat: 00-4333-57, Waltham, MA, USA). The cells were then washed in FACS buffer and stained with respective antibodies according to the manufacturer’s protocol: anti-mouse CD3 FITC (Cat: 100204) was purchased from BioLegend (San Diego, CA, USA), and anti-mouse CD4 PE (Cat: 12-0041-82) and CD8 Alexa Flour 700 (Cat: 56-0081-80) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). DAPI or PI staining was used for identifying dead cells.
The stained samples were analyzed using Gallios flow cytometer/CytoFLEX LX (Beckman Coulter, Brea, CA, USA), and the data were analyzed by Kaluza C analysis software (Beckman Coulter, Brea, CA, USA).
Mouse spleens were collected after sacrifice and dissociated by scalpel. A cell strainer (40 µm) was used to remove the debris. Isolated splenocytes were washed with PBS and subjected to RBC lysis using RBC lysis buffer (Thermo Fisher Scientific, Cat: 00-4333-57, Waltham, MA, USA). The cells were then washed in FACS buffer and stained with respective antibodies according to the manufacturer’s protocol: anti-mouse CD3 FITC (Cat: 100204) was purchased from BioLegend (San Diego, CA, USA), and anti-mouse CD4 PE (Cat: 12-0041-82) and CD8 Alexa Flour 700 (Cat: 56-0081-80) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). DAPI or PI staining was used for identifying dead cells.
The stained samples were analyzed using Gallios flow cytometer/CytoFLEX LX (Beckman Coulter, Brea, CA, USA), and the data were analyzed by Kaluza C analysis software (Beckman Coulter, Brea, CA, USA).
6
Apoptosis Detection in RPMI-2650 Cells
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After transfection for 48 h, 1 × 105 RPMI-2650 cells were treated with 5 μl of Annexin V and 5 μl of propidium iodide (PI) for 15 min in the dark at room temperature. Cell apoptosis was detected using an Annexin V-FITC cell apoptosis kit (130-092-052; Miltenyi Biotech, Waltham, MA, U.S.A.) and data were analyzed using Kaluza C Analysis Software (Beckman Coulter, Indianapolis, IN, U.S.A.).
7
Quantifying Murine T Cell Subsets by Flow Cytometry
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The isolated spleen cells from C57BL/6 J mice were subjected to flow cytometric analysis and then stained with the following fluorescence-conjugated antibodies: anti-Cluster of Differentiation 4 (CD4) antibody (FITC, ab218745, Abcam, Cambridge, UK), anti-CD25 antibody, (PE, 12–0251-82, Thermo Fisher Scientific), anti-Forkhead box P3 (Foxp3) antibody (Allophycocyanin (APC), ab200568, Abcam, UK) and anti-IL-17 antibody (Biotin, C48308-Biotin, Signalway Antibody, College Park, MD) at 4 °C for 1 h in the dark. The secondary antibodies included goat anti-rat IgG (Alexa Fluor® 488, ab150157, Abcam, UK) and goat anti-mouse IgG (Alexa Fluor® 647, ab150115, Abcam, UK). Then lymphocyte subsets in total CD4+ T cells were analyzed with flow cytometry in the dark. Changes on percentages of regulatory T cells (Treg cells, CD4+CD25+Foxp3+) and T helper 17 cells (Th17 cells, CD4+IL-17+) in total CD4+ T cells were measured with CytoFLEX Flow Cytometer (B96622, Beckman Coulter, Indianapolis, IN) in Kaluza C Analysis Software (Beckman Coulter).
8
Annexin V-FITC/PI Apoptosis Assay
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9HTE cell apoptosis was detected using flow cytometry with an Annexin V-FITC/propidium iodide (PI) apoptosis kit (A211; GeneBio Systems, Inc.) as per the manufacturer's instructions. Following transfection for 48 h, the 9HTE cells were harvested and then washed with cold phosphate-buffered saline (PBS) twice, followed by treatment with both Annexin V and PI (5 µl/well) for 20 min in the dark at room temperature. Cell apoptosis was further analyzed using a Guava easyCyte Benchtop Flow Cytometer (BR168323; Luminex Corporation) and Kaluza C Analysis Software (version 1.1.1, Beckman Coulter, Inc.).
9
Apoptosis Assessment of Meningioma Cells
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Forty eight hours after the transfection, the meningioma cells (1 × 10 5 ) were treated with Annexin V and propidium iodide (PI) together for 15 min at room temperature in the dark. Cell apoptosis was detected with Annexin V-FITC cell apoptosis kit (APOAF-50TST; Sigma-Aldrich, USA), and the data were analyzed using Kaluza C Analysis Software (Beckman Coulter, Indianapolis, IN, USA).
10
Annexin V-FITC/PI Apoptosis Assay
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For apoptosis detection, 1×105 HPAECs were treated with Annexin V (5 μL) and propidium iodide (PI) (5 μL) together for 15 minutes at room temperature in the dark. After cells were collected and washed with cold PBS twice, cell apoptosis was detected using an Annexin V-FITC/PI cell apoptosis kit (C1062L, Beyotime, China). Cell apoptosis was detected with Guava easyCyte Benchtop Flow Cytometer (BR168323; Luminex, Austin, TX) and data were analyzed using Kaluza C Analysis Software (version 2.1, Beckman Coulter, Indianapolis, IN).
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