Summit 6

Manufactured by Beckman Coulter

Sourced in United States

The Summit 6.2 software is a suite of data analysis tools designed for use with Beckman Coulter laboratory instruments. The software provides a platform for acquiring, processing, and managing data generated by Beckman Coulter products. Its core function is to enable users to analyze and interpret data from their laboratory experiments and assays.

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Lab products found in correlation

Moflo astrios cell sorter, by Beckman Coulter (2 mentions) Moflo astrio, by Beckman Coulter (2 mentions) 2 nbdg, by Thermo Fisher Scientific (2 mentions) Gene pulser 2 electroporation system, by Bio-Rad (1 mentions) Facsaria flow cytometer, by BD (1 mentions) Kaluza 1, by Beckman Coulter (1 mentions) Hoechst 33342, by Merck Group (1 mentions) Moflo astrios flow cytometer, by Beckman Coulter (1 mentions) Annexin 5 apc, by BioLegend (1 mentions) Lsr flow cytometer, by Beckman Coulter (1 mentions) Propidium iodide, by BioLegend (1 mentions) Moflo astrios eq, by Beckman Coulter (1 mentions) Moflo xdp, by Beckman Coulter (1 mentions) Annexin 5 fluorescein isothiocyanate fitc propidium iodide staining kit, by BD (1 mentions) Collagenase 5, by Merck Group (1 mentions)

Close spelling variants of this product

Software Summit Version 6.3.1.1

11 protocols using summit 6

Modulating UBE2A in BCR-ABL1+ Cells

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BA/F3_BCR-ABL1-positive cells were transfected with pMIGR1_UBE2A vectors (Online Supplementary Appendix) as by Puttini et al.^{14 (link)} and were analyzed for GFP positivity with a FACSAria flow cytometer (BD Bioscience, San Jose, USA) and FACS-sorted when transfection efficiency was lower than 85%.
32Dcl3-BCR/ABL1 cells were electroporated using a Gene Pulser® II Electroporation System (BIORAD) with pMIGR1_UBE2A WT and I33M vectors as described by Puttini et al.^{14 (link)} To obtain stable UBE2A WT or I33M cell lines, GFP positive population was FACS-sorted with a MoFlo Astrios cell sorter equipped with Summit 6.3 software (both from Beckman Coulter, Miami, FL, USA).
For UBE2A silencing, K562 cells were infected with lentivirus obtained from MISSION-shRNA pLKO.1-based vectors (TRCN0000320625) (Sigma-Aldrich, Missouri, USA) and packaged using 293FT cell line. As a control, a pLKO.1MISSION non-target control vector (SHC002) (Sigma-Aldrich) was used. After infection K562 cells were maintained in 2 μg/mL puromycin for selection of silenced (K562_shUBE2A) and control cells (K562_shNC).

Magistroni V., Mauri M., D’Aliberti D., Mezzatesta C., Crespiatico I., Nava M., Fontana D., Sharma N., Parker W., Schreiber A., Yeung D., Pirola A., Readelli S., Massimino L., Wang P., Khandelwal P., Citterio S., Viltadi M., Bombelli S., Rigolio R., Perego R., Boultwood J., Morotti A., Saglio G., Kim D.W., Branford S., Gambacorti-Passerini C, & Piazza R. (2019). De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways. Haematologica, 104(9), 1789-1797.

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Quantifying Human Cell Engraftment in Mice

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Human cells engraftment was check every 15 days by flow cytometry. For the blood collection, mice were placed under a heat lamp to promote peripheral vasodilatation and were mechanically restrained using a plexiglass chamber. Then, a small transverse cut was performed in the lateral tail vein with a sterile lancet. Blood drops were collected with a micropipette and mixed with 10 μl EDTA 0.5 M. A maximum volume of 100 μl of blood was collected. After blood collection, a slight pressure was applied to ensure the bleeding stop. 100 μl EDTA-anticoagulated peripheral blood were lysed twice in red blood cells lysis buffer (140 mM NH₄Cl, 8 mM Tris, pH 7.2) at RT for 5 minutes, and then cells were washed and suspended in PBS. Subsequently, cells were stained with the Alexa Fluor® 700-conjugated anti-human CD45 (HI30; Biolegend, San Diego, CA, USA) and PE-conjugated anti-mouse CD45 (30-F11; Biolegend) antibodies, at RT for 30 minutes. Dual-color flow cytometry was performed on MoFlo Astrios cell sorter equipped with Summit 6.3 software (both from Beckman Coulter, Miami). The acquisition process was stopped when at least 5000 events were collected in the population gate. Off-line analysis was performed using Kaluza 1.3 software (Beckman Coulter). Engraftment occurred in 70% of mice.

Fontana D., Ramazzotti D., Aroldi A., Redaelli S., Magistroni V., Pirola A., Niro A., Massimino L., Mastini C., Brambilla V., Bombelli S., Bungaro S., Morotti A., Rea D., Stagno F., Martino B., Campiotti L., Caocci G., Usala E., Merli M., Onida F., Bregni M., Elli E.M., Fumagalli M., Ciceri F., Perego R.A., Pagni F., Mologni L., Piazza R, & Gambacorti-Passerini C. (2020). Integrated Genomic, Functional, and Prognostic Characterization of Atypical Chronic Myeloid Leukemia. HemaSphere, 4(6), e497.

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Testicular Germ Cell Analysis and Sorting

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Cell analysis and sorting was carried out on WT and Ankrd31^–/– testicular germ cell populations (n = 6 animals for each genotype), using a MoFlo Astrios^EQ high speed cell sorter (Beckman Coulter Inc., Brea, CA, United States) with a nozzle of 90 μm using Summit 6.3 software (Beckman Coulter Inc.). Analysis was triggered with a blue laser (488 nm), debris was eliminated based on morphological criteria using forward scatter (FSC) vs. side scatter (SSC). We used Hoechst 33342 (Sigma) fluorophore to measure DNA content, excited using a violet laser (405 nm), and the blue fluorescence emitted was measured trough a 448/59 nm band pass filter. To preserve cell quantity for post-sorting experiences, a maximum of 200,000 events was analyzed before proceeding to the gating selection. Three peaks with increasing Hoechst fluorescence, representative of haploid (1C—Region 1), diploid (2C—Region 2), and tetraploid (4C—Region 3) cells, were detected and sorted using a dot-plot of side scatter (SSC) vs. Hoechst blue fluorescence. Sorting flow rate was adapted to the sample and set around 10,000 events/s for the most concentrated samples; for cell recovery, a 1.5 ml Eppendorf tube containing 350 μl of RPMI with 10% FBS was used.

Manfrevola F., Martinez G., Coutton C., Rocco D., Reynaud K., Le Vern Y., Froment P., Beauclair L., Aubert D., Pierantoni R., Chianese R, & Guillou F. (2021). Ankrd31 in Sperm and Epididymal Integrity. Frontiers in Cell and Developmental Biology, 9, 741975.

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Bacterial Purification and Flow Cytometry

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Bacteroids were purified as described^{24 (link)}. Bacteria were then fixed by heat treatment (70 °C, 10 min) and stained by DAPI (20 µg/ml) before flow cytometry analysis using a MoFlo ASTRIOS flow cytometer (Beckman Coulter). The bacterial DNA content was assessed by the DAPI fluorescence with a 355-nm laser line. Each single event was recorded and analyzed with the Summit 6.2 software (Beckman Coulter).

Barrière Q., Guefrachi I., Gully D., Lamouche F., Pierre O., Fardoux J., Chaintreuil C., Alunni B., Timchenko T., Giraud E, & Mergaert P. (2017). Integrated roles of BclA and DD-carboxypeptidase 1 in Bradyrhizobium differentiation within NCR-producing and NCR-lacking root nodules. Scientific Reports, 7, 9063.

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Flow Cytometry Analysis of Bacteroid Sizes

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For each condition, nodules were harvested on more than 15 inoculated plants and pooled to have a representative sample for flow cytometry analyses. Nodules were crushed with a mortar and a pestle in a bacteroid extraction buffer (BEB: 125 mM KCl, 50 mM Na-succinate, 50 mM TES, 0.1% BSA, pH 7.0). The nodule homogenate was centrifuged for 10 min at 100g to pellet plant debris. The supernatant containing the bacteroids was centrifuged for 10 min at 3600g to pellet the bacteroids before resupsension in a small volume of BEB. All the procedures were performed at 4°C. Bacteroids were heat-killed (for 10 min at 70°C) and stained using 50 μg ml⁻¹ propidium iodide (PI). Cell size (forward scatter of the laser ray) and DNA content (level of fluorescence of the PI) were determined on a MoFlo Astrios flow cytometer and the results were analyzed using the Summit 6.2 software (Beckman Coulter).

Lamouche F., Bonadé-Bottino N., Mergaert P, & Alunni B. (2019). Symbiotic Efficiency of Spherical and Elongated Bacteroids in the Aeschynomene-Bradyrhizobium Symbiosis. Frontiers in Plant Science, 10, 377.

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Apoptosis Induction by Aspirin and CDDP

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Cells were plated in triplicate in a six-well plate at a density of 1 ×10⁵ cells/well and incubated for 24 h. Then, cells were treated with different concentrations of aspirin (0 µM, 100 µM, and 1,000 µM) and/or CDDP (20 µM). After a 48-h incubation at 37 °C, cells were harvested, trypsinized and washed with cold phosphate-buffered saline (PBS). Subsequently, cells were stained with APC Annexin V and propidium iodide (PI) (BioLegend, Inc., San Diego, CA, USA). The stained cells were immediately analyzed using an LSR flow cytometer (Beckman Coulter, Inc., 250 S. Kraemer Boulevard, Brea, CA, USA), and the data were analyzed using Summit 6.2 software (Beckman Coulter, Inc., 250 S. Kraemer Boulevard, Brea, CA, USA).

Guo J., Zhu Y., Yu L., Li Y., Guo J., Cai J., Liu L, & Wang Z. (2021). Aspirin inhibits tumor progression and enhances cisplatin sensitivity in epithelial ovarian cancer. PeerJ, 9, e11591.

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Chromatin Fragmentation and Flow Cytometry

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Fragmented chromatin particles were collected and resuspended in the sorting buffer (2 saline-sodium citrate, 20% (vol/vol) formamide and 0.2mg/ml RNase-free BSA in nuclease-free water) and detected based on SSC (side scatter) and FSC (forward scatter) under 488nm excitation using the MoFlo Astrios EQ flow cell sorter (Beckman Coulter). Latex beads measuring 100, 200, and 300nm from the Photon Correlation Spectroscopy control mixed kit (Beckman Coulter, 6602336) were used as the standard size particles, and the sorting buffer was used as the background control. The data was collected and analyzed by the Summit 6.2 software (Beckman Coulter), and the gates were set based on the standard particle sizes. The measurements were done in 3 biological replicas with one shown in Fig. 3c. On average, ~250 and ~ 480 thousand particles were measured for the buffer-alone control and the fragmented chromatin samples respectively.

Cao H., Xu D., Cai Y., Han X., Tang L., Gao F., Qi Y., Cai D., Wang H., Ri M., Antonets D., Vyatkin Y., Chen Y., You X., Wang F., Nicolas E, & Kapranov P. (2021). Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans. BMC Biology, 19, 108.

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Metformin Induces Ovarian Cancer Apoptosis

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The effects of metformin on ovarian cancer cell apoptosis were assessed by flow cytometry using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining kit (BD Pharmingen; BD Biosciences, San Jose, CA, USA). As cells transduced with lenti-virus carried green fluorescence, their apoptosis rates were assessed by using Annexin V-phycoerythrin (PE)/7-aminoactinomycin D (7-AAD) staining kit (BD Pharmingen; BD Biosciences). After 24 h of exposure to metformin, cells were washed twice with ice-cold PBS, re-suspended in 500 µl binding buffer, and stained with 5 µl FITC-conjugated 5 µl Annexin V or 5 µl Annexin V-PE conjugated and 7 µl 7-AAD, following gentle mixing, cells were incubated at room temperature shielded from light for 15 min. Following washing with binding buffer, flow cytometry analysis was performed using a flow cytometry sorting system (MoFlo XDP) with Summit 6.2 software (both from Beckman Coulter, Inc., Brea, CA, USA). Each assay was run at least three times.

Tang G., Guo J., Zhu Y., Huang Z., Liu T., Cai J., Yu L, & Wang Z. (2018). Metformin inhibits ovarian cancer via decreasing H3K27 trimethylation. International Journal of Oncology, 52(6), 1899-1911.

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Pancreatic Single-Cell Isolation and Analysis

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Pancreatic single-cell suspensions were prepared as described⁷⁹ (link) by mincing the tissue and then digesting in 1 mg/mL collagenase V (Sigma-Aldrich) at 37°C for 15–20 minutes. Digested samples were filtered through a 40-μm strainer and subjected to a red blood cell lysis step. Samples were submitted for flow analysis in Hank’s balanced salt solution with 2% fetal bovine serum. Antibodies were used in combinations of the following: CD45-Pacific Orange (1:50; BD Pharmingen, Franklin Lakes, NJ), PDGFR⍺-Phycoerythrin (1:50; BD Pharmingen), and PDGFR⍺-Phycoerythrin-Cy7 (1:50; BD Pharmingen). Fluorescence-activated cell sorting was performed on a MoFlo Astrios (Beckman Coulter) and data were analyzed using Summit 6.1 Software (Beckman Coulter, Brea, CA) and FlowJo 10.6 (FlowJo, Ashland, OR). The gating strategy was as follows: cells were first selected on forward (FSC-Area) and side (SSC-Area) scatters to exclude debris. Then single cells were gated based on SSC-Height vs SSC-Width and FSC-Height vs FSC-Width parameters. Alive cells were selected from an SSC-Height vs 4′,6-diamidino-2-phenylindole plot. Nonimmune cells were selected from an SSC-Height vs CD45-Pacific Orange plot. At least 1 × 10⁵ events that pass these selection parameters were recorded for each sample. Further gating based on PDGFR, YFP, and Tomato parameters was used to analyze and sort the cells.

Garcia P.E., Adoumie M., Kim E.C., Zhang Y., Scales M.K., El-Tawil Y.S., Shaikh A.Z., Wen H.J., Bednar F., Allen B.L., Wellik D.M., Crawford H.C, & Pasca di Magliano M. (2020). Differential Contribution of Pancreatic Fibroblast Subsets to the Pancreatic Cancer Stroma. Cellular and Molecular Gastroenterology and Hepatology, 10(3), 581-599.

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Glucose Uptake Assay in Promastigotes

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Promastigotes (5 × 10⁶ protozoa/mL) were treated with ¹/₅ IC₅₀/4 h NaNO₂ in HBSS at 25 °C for 4 h. After that, protozoa were washed with PBS and incubated with 300 µM 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) (2-NBDG, Molecular Probes, Eugene, OR, USA), a fluorescent glucose analogue, for 30 min. The 2-NBDG specificity was monitored by the parasite incubation at 4 °C for the same amount of time, to decrease the compound uptake. 2-NBDG+ parasites were analyzed using a CytekDxP multi-color upgrade flow cytometer (Cytek, Fremont, CA, USA). A total of 10,000 events were acquired in the region previously established by protozoa morphology, and analyses were performed in Summit 6.1 software (Beckman Coulter, Brea, CA, USA).

Pinho N., Bombaça A.C., Wiśniewski J.R., Dias-Lopes G., Saboia-Vahia L., Cupolillo E., de Jesus J.B., de Almeida R.P., Padrón G., Menna-Barreto R, & Cuervo P. (2022). Nitric Oxide Resistance in Leishmania (Viannia) braziliensis Involves Regulation of Glucose Consumption, Glutathione Metabolism and Abundance of Pentose Phosphate Pathway Enzymes. Antioxidants, 11(2), 277.

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