Kaluza analysis software 2

Manufactured by Beckman Coulter

Sourced in United States, Germany

Kaluza Analysis Software 2.1 is a flow cytometry data analysis platform developed by Beckman Coulter. The software provides advanced data visualization and analysis tools for researchers to interpret complex flow cytometry datasets.

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Lab products found in correlation

Cytoflex, by Beckman Coulter (4 mentions) Cxp software, by Beckman Coulter (3 mentions) Cytoflex flow cytometer, by Beckman Coulter (3 mentions) Cytoflex s, by Beckman Coulter (2 mentions) Navios flow cytometer, by Beckman Coulter (2 mentions) Dna prep reagents kit, by Beckman Coulter (2 mentions) Bapta am, by Thermo Fisher Scientific (1 mentions) Fluo 3 am, by Thermo Fisher Scientific (1 mentions) Annexin 5 binding buffer, by BioLegend (1 mentions) Annexin 5, by BioLegend (1 mentions) Cytoflex lx, by Beckman Coulter (1 mentions) Zombie nir, by BioLegend (1 mentions) Catalase solution, by Merck Group (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole dapi, by BioLegend (1 mentions) Cytoflex s flow cytometer, by Beckman Coulter (1 mentions) Prism 9, by GraphPad (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by BD (1 mentions) Rnase a, by EURx (1 mentions)

29 protocols using kaluza analysis software 2

Apoptosis and Proliferation Assay of BCR-ABL1 Transformed Cells

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Cells (1×10⁶) were treated with imatinib (0, 3, 6 and 9 µM), and after 24 h, the cells were collected and washed twice with cold PBS, and incubated for 20 min at room temperature in 1:20 diluted PE-conjugated Annexin V and anti-7-aminoactinomycin D (7-AAD) (cat. no. 559763; BD Biosciences), according to the manufacturer's instructions. Then, the cells were analyzed by flow cytometer (Beckman Coulter, Inc.). The Aid KO and Aid WT BCR-ABL1-transformed pro-B cells were starved in FBS-free medium for 24 h, and measured for apoptosis by flow cytometry. Flow cytometric data were analyzed with Kaluza Analysis Software 2.1 (Beckman Coulter, Inc.).
Proliferation assays were performed using the BrdU kit (cat. no. 552598; BD Biosciences) according to the manufacturer's instructions. Briefly, a population of 1×10⁶ cells were cultured in 6-well plates and labeled with BrdU labeling reagent for 45 min before harvesting. The cells were centrifuged at 300 × g for 5 min at 4°C, then fixed and permeabilized with Cytofix/Cytoperm buffer and Cytoperm permeabilization buffer plus. Next, cells were treated with DNase for 1 h at 37°C and further incubated with APC-conjugated anti-BrdU for 20 min at room temperature. BrdU incorporation was measured using a flow cytometer (Beckman Coulter, Inc.). Flow cytometric data were analyzed with Kaluza Analysis Software 2.1 (Beckman Coulter, Inc.).

Zhang P., Wang Y., Qin M., Li D., Odhiambo W.O., Yuan M., Lv Z., Liu C., Ma Y., Dong Y, & Ji Y. (2020). Involvement of Blnk and Foxo1 in tumor suppression in BCR-ABL1-transformed pro-B cells. Oncology Reports, 45(2), 693-705.

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Measuring Calcium Flux and NFAT Activation

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To measure the calcium flux after plasma treatment, TK6 cells were loaded with Fluo-3-AM (Thermo Fisher Scientific, Germany) for 15 min according to the manufacturer's instructions. JE6.1-TPR were not used for this experiment because their reporter signal would have interfered with the calcium-induced Fluo-3 signal. Afterward, cells were resuspended in DMEM10 and treated with the micro plasma jet for exposure various times. PMA (20 ng/mL) and Ionomycin (1 μg/mL) were used as a positive control. 10 min or 1 h after plasma treatment the Fluo-3 signal was measured using flow cytometry (CytoFLEX S; Beckman-Coulter, Germany), and data analysis was performed using Kaluza analysis software 2.1.3 (Beckman-Coulter, Germany). To analyze the impact of calcium on the activation of the transcription factor NFAT in JE6.1-TPR, cells were loaded with the calcium chelator BAPTA-AM (Thermo Fisher Scientific, Germany) according to the manufacturer's instructions before plasma treatment. 18 h after plasma treatment, the activation of NFAT was measured by its reporter signal using flow cytometry.

Mrochen D.M., Miebach L., Skowski H., Bansemer R., Drechsler C.A., Hoffmann U., Hein M., Mamat U., Gerling T., Schaible U., von Woedtke T, & Bekeschus S. (2022). Toxicity and virucidal activity of a neon-driven micro plasma jet on eukaryotic cells and a coronavirus. Free Radical Biology & Medicine, 191, 105-118.

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Cell Viability Assessment by Flow Cytometry

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If not indicated otherwise, cell viability was determined 18 h after plasma treatment by staining with Annexin V and DAPI (4′,6-Diamidino-2-phenylindole) or Zombie NIR (all BioLegend, The Netherlands). After incubation, cells were harvested either with (HaCaT, Calu-3) or without (JE6.1-TPR, THP-1-R) Accutase. Care was taken to include cells in the supernatant of adherent cells (HaCaT, Calu-3) to avoid missing dying or dead cells that had already lost adhesion. Afterward, cells were washed with Annexin V Binding Buffer (BioLegend, The Netherlands) and stained with either Annexin V (Alexa Fluor 647) and DAPI (1 μM) (HaCaT, Calu-3), Annexin V (PE) and DAPI (1 μM) (THP-1-R), or Annexin V (Alexa Fluor 647) and Zombie NIR (JE6.1-TPR). Cells were measured using flow cytometry (CytoFLEX LX; Beckman-Coulter, Germany), and data analysis was performed using Kaluza analysis software 2.1.3 (Beckman-Coulter, Germany). Viable cells were defined as Annexin V⁻ and Zombie NIR⁻ or DAPI⁻.

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Microorganism Enumeration by Flow Cytometry

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Following gas plasma exposure, the microorganisms in the microwell plates were kept at room temperature in the dark for 16 h. In some conditions, 2 µL of catalase solution (Sigma-Aldrich, Taufkirchen, Germany) was added (final concentration: 20 µg/mL) either before or after gas plasma exposure of samples. Then, to each well, fixative was added (4% paraformaldehyde; Sigma-Aldrich) and incubated for 10 min in the dark. Then, to each well, 4′,6-diamidino-2-phenylindole (DAPI, final concentration 10 µM; BioLegend, Amsterdam, The Netherlands) was added, and the microorganisms were incubated for 15 min in the dark. All microorganisms contain ample amounts of DNA to which DAPI binds and becomes fluorescent. This way, microorganisms can be conveniently detected using flow cytometry. Next, the microplates were added to an autosampler of a CytoFLEX S flow cytometer (Beckman-Coulter, Krefeld, Germany) trigger through the forward scatter (488 nm laser diode) and collection of DAPI fluorescence via λ_ex 405 nm and λ_em 450 ± 45 nm. After mixing by the autosampler, 100µL of cell suspension was acquired from each well. The resulting .fcs (3.1 standard) data files were analyzed using Kaluza analysis software 2.1.3 (Beckman-Coulter).

Clemen R., Singer D., Skowski H, & Bekeschus S. (2023). Argon Humidification Exacerbates Antimicrobial and Anti-MRSA kINPen Plasma Activity. Life, 13(2), 257.

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Flow Cytometry Data Analysis

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Flow cytometry samples were quantitively analyzed in this study using Kaluza analysis software 2.1.3 (Beckman-Coulter). Data normalization was done using Excel 2021 (Microsoft, Redmond, WA, USA). Data analysis and graphing were done utilizing prism 9.4.1 (GraphPad Software, San Diego, CA, USA). One-way ANOVA with Turkey’s multiple comparison test or t-test was executed to determine the degree of statistical significance between groups. The level of significance is indicated as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Wolff C.M, & Bekeschus S. (2022). Synergistic In Vitro Anticancer Toxicity of Pulsed Electric Fields and Glutathione. International Journal of Molecular Sciences, 23(23), 14772.

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Cell Cycle Analysis by Flow Cytometry

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MOLT-4 cells were prepared as described for the MMP assay, except that after cells were incubated for 24, 48, and 72 h with analyzed reagents, cells were fixed in cold 70% ethanol for 30 min. Fixed cells were stained for 15 min in buffer containing 500 µg/mL RNAse A (EURx, Gdansk, Poland) and 2.5 µg/mL of DAPI (Merck, Darmstadt, Germany). The fluorescence of DAPI was measured at excitation λ_ex = 355 nm and emission λ_em = 440 nm with a flow cytometer (LSR II, BD Biosciences, Mountain View, CA, USA). Each experiment was performed in triplicate. Data were analyzed off-line using the Kaluza Analysis Software 2.1.3 (Beckman Coulter Life Sciences, IN, USA). The assay was based on the method described in Żołnowska et al. [110 (link)].

Koszałka P., Stasiłojć G., Miękus-Purwin N., Niedźwiecki M., Purwin M., Grabowski S, & Bączek T. (2022). The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro. International Journal of Molecular Sciences, 23(9), 4753.

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Flow cytometric analysis of P3 IFP-MSC

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Flow cytometric analysis was performed on P3 IFP-MSC (n = 3). Briefly, 2.0 × 10⁵ cells were labelled with CD10 monoclonal antibody (Biolegend, San Diego, CA, USA) and the corresponding isotype control. All samples included a ghost red viability dye (Tonbo Biosciences, San Diego, CA, USA). Data (20.000 events collected) were acquired using a Cytoflex S (Beckman Coulter, Brea, CA, USA) and analyzed using Kaluza analysis software 2.1. (Beckman Coulter).

Kouroupis D., Kaplan L.D., Huard J, & Best T.M. (2023). CD10-Bound Human Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles Possess Immunomodulatory Cargo and Maintain Cartilage Homeostasis under Inflammatory Conditions. Cells, 12(14), 1824.

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Transduction of BCR-ABL1-Expressing Cells

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MSCV vector co-expressing human BCR-ABL1 (p210) and hCD4 (MSCV-BCR-ABL1-IRES-hCD4) and MSCV vector expressing hCD4 have been previously described (36 (link)). 293T cells were transfected with MSCV vectors and PKAT2 package vector using X-tremeGENE HP DNA transfection reagent (Roche Diagnostics) at 37°C. After 48 h of transfection, viral supernatants were collected, filtered, and stored at −80°C.
BCR-ABL1-transformed pro-B cells and the control cells were obtained by transduction of D345 cells with pMSCV-BCR-ABL1-hCD4 or pMSCV-EV-hCD4 viral supernatants with 1 µg/ml polybrene; then the six-well dishes were spun at 1,000 × g for 1.5 h at room temperature. The medium was changed after 4 h and incubated at 37°C. After 72 h, the cells were incubated with APC-conjugated anti-human CD4 antibody (cat. no. 561840; BD Biosciences) at a 1:10 dilution for 20 min at 4°C, and sorted by BD FACSAria II (BD Biosciences). Flow cytometric data were analyzed with Kaluza Analysis Software 2.1 (Beckman Coulter, Inc.).

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Platelet P-selectin Expression by Flow Cytometry

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Platelet surface expression of P-selectin (CD62P) was evaluated using an APC-conjugated anti-CD62P monoclonal antibody (2 μg/ml, mouse IgG1κ; eBioscience, San Diego, CA) or isotype control antibody (eBioscience). Collagen (2 μg/ml; ChronoLog) was used as platelet agonist. Platelets were analyzed by flow cytometry using a BD Accuri C6 Plus (BD Biosciences, San Jose, CA) flow cytometer and Kaluza Analysis Software 2.1 (Beckman Coulter, MD). Platelets were gated based on their characteristic scatter properties.

Boone B.A., Murthy P., Miller-Ocuin J.L., Liang X., Russell K.L., Loughran P., Gawaz M., Lotze M.T., Zeh HJ I.I.I, & Vogel S. (2019). The platelet NLRP3 inflammasome is upregulated in a murine model of pancreatic cancer and promotes platelet aggregation and tumor growth. Annals of hematology, 98(7), 1603-1610.

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Cell Cycle and Cell Death Analysis

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For cell cycle and cell death analyses, cells were trypsinized and incubated in cold PBS supplemented with 5% fetal calf serum (Sigma-Aldrich; PBS-FACS). DNA was stained either by propidium iodide (PI) or by Hoechst. For PI staining, cells were fixed in cold 70% ethanol, added dropwise while vortexing, and incubated on ice for 30 minutes. Cells were centrifuged and pellets were washed twice with PBS-FACS. 50 μl RNase A solution (100 μg/ml in PBS) was added to the pellet, followed by staining with 400 μl PI solution (50 μg/ml in PBS) per million cells. Cells were incubated for 10’ at 25°C. For Hoechst staining, pellets were incubated in the dark with 10 mg/ml Hoechst 33358 for 15’ at 4°C. Data acquisition was performed using the CytoFLEX flow cytometer (Beckman Coulter) or the BD FacsJAZZ cell sorter (BD Biosciences). Data analysis was performed using the Kaluza Analysis software 2.1 (Beckman Coulter). Gating strategy: An SSC-A/FSC-A gate was set in order to exclude cell debris, and an FSC-A/FSC-H gate was then set in order to exclude doublets. Cell cycle phases were determined manually using linear gating based on the 2N and 4N peaks of the histogram. Cell death was assessed by quantifying the fraction of cells in the subG1 population, and mitotic arrest was assessed by quantifying the fraction of cells in the G2/M population. Gating strategy is shown in Supplementary Fig. 3.

Cohen-Sharir Y., McFarland J.M., Abdusamad M., Marquis C., Bernhard S.V., Kazachkova M., Tang H., Ippolito M.R., Laue K., Zerbib J., Malaby H.L., Jones A., Stautmeister L.M., Bockaj I., Wardenaar R., Lyons N., Nagaraja A., Bass A.J., Spierings D.C., Foijer F., Beroukhim R., Santaguida S., Golub T.R., Stumpff J., Storchova Z, & Ben-David U. (2021). Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition. Nature, 590(7846), 486-491.

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