Solution 5

Manufactured by Lonza

Sourced in Germany

Solution V is a high-performance laboratory centrifuge designed for a wide range of applications. It features a compact and robust construction, providing reliable and consistent performance. The centrifuge is capable of handling a variety of sample volumes and can achieve high rotational speeds, making it suitable for common laboratory procedures such as sample preparation, cell separation, and biomolecule purification.

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Lab products found in correlation

Amaxa nucleofector apparatus, by Lonza (2 mentions) Amaxa nucleofector 2 device, by Lonza (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Amaxa nucleofector device, by Lonza (1 mentions) Plv hu6 sgrna hubc dcas9 krab t2a gfp, by Addgene (1 mentions) Sh800 cell sorter, by Sony (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Nk cell isolation kit 2, by Miltenyi Biotec (1 mentions) Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Nk cell isolation kit, by Miltenyi Biotec (1 mentions) Rpmi 1640, by Corning (1 mentions) Nucleofector device, by Lonza (1 mentions) Lymphocyte separation medium, by Corning (1 mentions) Ingenio electroporation solution, by Mirus Bio (1 mentions) Amaxa nucleoporation, by Lonza (1 mentions) Sigenome smartpool, by Horizon Discovery (1 mentions)

8 protocols using solution 5

siRNA Knockdown in CLL Cells

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siRNA against human HSP90, BTK or AKT (2 μg) were transfected into CLL cells using Amaxa Nucleofector II device (Amaxa, Cologne, Germany) and Solution V (Amaxa). Nucleofector program U-15 was applied according to the manufacturer’s instructions.^{28 (link),29 ,40 (link),41 (link)}

Guo A., Lu P., Lee J., Zhen C., Chiosis G, & Wang Y. (2017). HSP90 stabilizes B-cell receptor kinases in a multi-client interactome: PU-H71 induces CLL apoptosis in a cytoprotective microenvironment. Oncogene, 36(24), 3441-3449.

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Ex Vivo Functional Analysis of PTCL CD30+ Cells

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A human PTCL CD30+ cell line (Fe-Pd) was used to perform the ex vivo functional experiments. Transient transfections were performed by nucleofection, using an Amaxa apparatus, program X-01 and solution V (Amaxa, Cologne, Germany). Total RNA from Fe-Pd cell line was extracted with Trizol according to the manufacturer's instructions (Invitrogen, Life Technologies). RNA was quantified using ND-1000 spectrophotometer running software version 3.0.1 (NanoDrop Technologies Inc.).
Gene expression profiling was executed on transfected and untransfected Fe-Pd cells using Gene Chip 2.0 Whole Transcript Assay (Affymetrix, Santa Clara, CA, USA) (details are provided in Supplementary File).

Laginestra M.A., Piccaluga P.P., Fuligni F., Rossi M., Agostinelli C., Righi S., Sapienza M.R., Motta G., Gazzola A., Mannu C., Sabattini E., Bacci F., Tabanelli V., Sacchetti C.A., Barrese T.Z., Etebari M., Melle F., Clò A., Gibellini D., Tripodo C., Inghirami G., Croce C.M, & Pileri S.A. (2014). Pathogenetic and diagnostic significance of microRNA deregulation in peripheral T-cell lymphoma not otherwise specified. Blood Cancer Journal, 4(11), 259-.

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siRNA Knockdown of Borcs and Kif Genes in Macrophages

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The siRNA-mediated gene knockdown was carried out as described previously^{50 (link)}. Briefly, Raw264.7 macrophages and BMDMs were resuspended in 90 µL of solution V (for Raw264.7 cells; Lonza) or solution for mouse macrophages (for BMDMs; Lonza), respectively. The cell suspension was mixed with either 1.5 μg of scrambled siRNAs or siRNAs against Borcs5, Borcs6, Borcs7, Borcs8, Kif5a, Kif5b and Kif1b, and then transferred to a nucleofection cuvette and nucleofected using the Amaxa Nucleofector device (Amaxa Biosystems) set to either programme D-032 (for RAW264.7 macrophages) or Y-001 (for BMDMs). At 24 h post-transfection, the cells were collected and plated in preparation for the experiments.

Tunganuntarat J., Kanjanasirirat P., Khumpanied T., Benjaskulluecha S., Wongprom B., Palaga T., Siregar T.A., Borwornpinyo S., Chaiprasert A., Palittapongarnpim P, & Ponpuak M. (2023). BORC complex specific components and Kinesin-1 mediate autophagy evasion by the autophagy-resistant Mycobacterium tuberculosis Beijing strain. Scientific Reports, 13, 1663.

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Nox2 and Sod2 Knockdown in RAW264.7 Macrophages

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siRNA-mediated knockdown was performed as previously described (Ponpuak et al. 2009 (link)). In brief, RAW264.7 macrophages were collected and resuspended in 90 μl of solution V (Lonza). Scramble control siRNAs or siRNAs against Nox2 or Sod2 was added to the cell suspension, transferred to a nucleofection cuvet, and nucleofected using the Amaxa Nucleofector apparatus (Amaxa Biosystems; program D-032). At 24 h post-transfection, cells were collected and plated for assays.

Siregar T.A., Prombutara P., Kanjanasirirat P., Kunkaew N., Tubsuwan A., Boonmee A., Palaga T., Khumpanied T., Borwornpinyo S., Chaiprasert A., Utaisincharoen P, & Ponpuak M. (2022). The autophagy-resistant Mycobacterium tuberculosis Beijing strain upregulates KatG to evade starvation-induced autophagic restriction. Pathogens and Disease, 80(1), ftac004.

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CRISPR-Mediated Gene Regulation in HEK293 Cells

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pLVhU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP was purchased from Addgene (Cambridge). Oligos encoding sgRNAs designed using GPP sgRNA Designer (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design-crisprai) were annealed and cloned into the plasmid. HEK293 cells were washed twice with PBS and 2 × 10⁶ cells were resuspended in 100 µL of Solution V (Lonza) and then mixed with 5 µg purified sgRNA expression vector or the empty vector as a negative control before nucleofection using program Q-001 of a Nucleofector 2b (Lonza). To enrich transfectants, single cells were sorted 16 h post-nucleofection using an SH800 cell sorter (SONY), and GFP-positive cells were enriched. Cells were then cultured in complete Dulbecco's modified Eagle's medium in two poly-l-lysine-coated wells of a 24-well plate at 37°C in a humidified atmosphere with 5% CO₂ for 16 h before treatment with 200 mM sorbitol for 3 h.

Iwakiri J., Tanaka K., Chujo T., Takakuwa H., Yamazaki T., Terai G., Asai K, & Hirose T. (2023). Remarkable improvement in detection of readthrough downstream-of-gene transcripts by semi-extractable RNA-sequencing. RNA, 29(2), 170-177.

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siRNA-Mediated Knockdown in Macrophages

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siRNA-mediated knockdown was performed as previously described^{38 (link)}. In brief, Raw264.7 macrophages or BMDMs were harvested and re-suspended in 90 µL of solution V (for RAW264.7 cells; Lonza) or solution for mouse macrophages (for BMDMs; Lonza). Scrambled siRNAs or siRNAs against Kxd1 or Plekhm2 (Dhamacon; 1.5 µg/reaction) were added to the cell suspension, transferred to an electroporation cuvette and nucleofected using the Amaxa Nucleofector apparatus (Amaxa Biosystems) with program D-032 (for RAW264.7 macrophages) or Y-001 (for BMDMs). At 24 h after transfection, cells were harvested and plated for assays.

Laopanupong T., Prombutara P., Kanjanasirirat P., Benjaskulluecha S., Boonmee A., Palaga T., Méresse S., Paha J., Siregar T.A., Khumpanied T., Borwornpinyo S., Chaiprasert A., Utaisincharoen P, & Ponpuak M. (2021). Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain. Scientific Reports, 11, 4342.

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PBMC Isolation and NK Cell Purification

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Peripheral blood mononuclear cells (PBMCs) were enriched by density gradient centrifugation using Lymphocyte Separation Medium (Corning Life Sciences.) Cells were maintained in RPMI 1640 (Corning Life Sciences) supplemented with 10% FBS (Gibco), 100 mg/ml streptomycin (Gibco), and 100U/ml penicillin (Gibco). For some experiments, NK cells were isolated from PBMCs using NK Cell Isolation Kit (Miltenyi). In the case of NKL cell line, the medium was supplemented with 50U/ml of recombinant interleukin-2 (IL-2, Peprotech). Mouse NK cells were obtained from splenocytes, purified using NK Cell Isolation Kit II (Miltenyi) and cultured with additional 50μM of β-Mercaptoethanol, and 500U/ml of recombinant IL-2. Cells were maintained at 37°C and 5% CO2. NKL cells were electroporated with 100nM of CD3ζ siRNA or control (Dharmacon) using a nucleofector device (Lonza). Briefly, 3×106 cells were washed twice with PBS, resuspended in 100μl of Solution V (Lonza) and electroporated using the program O-017. After electroporation, cells were placed in prewarmed medium and cultured for 72h.

Suárez-Fueyo A., Bradley S.J., Katsuyama T., Solomon S., Katsuyama E., Kyttaris V.C., Moulton V.R, & Tsokos G.C. (2018). Downregulation of CD3ζ in Natural Killer Cells from Systemic Lupus Erythematosus Patients Confers a Pro-Inflammatory Phenotype. Journal of immunology (Baltimore, Md. : 1950), 200(9), 3077-3086.

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Optimization of Protein Knockdown in Skin Cells

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NHEKs and SCC9s were electroporated with siRNA oligonucleotides at a final concentration of 10 nM via AMAXA nucleoporation (Lonza) using the Ingenio Electroporation Solution (Mirus) or solution V (Lonza) and program X-001. siRNA directed towards DSG1 (5' CCA UUA GAG AGU GGC AAU AGG AUG A), DP (Invitrogen Pool: oligonucleotides: 5’-GAA GAG AGG UGC AGG CGU A, 5’ GAC CGU CAC UGA GCU AGU A, and 5’ AAA CAG AAC GCU CCC GAU A), and COPS3 (Invitrogen Stealth siRNA_siCOPS3-465: 5’ AUC AAU GUU AUG AAG CAU GGC UGG G or Integrated DNA Technologies Pool_IDT Pool: 5’-GGA UAU CUG UAA AGA GAA UGG AGC C, 5’-GGA UGUACA AGA ACA CUC CUU GGG C, 5’-UCC AUC CUG AGC UAA ACA AGA GAA A, and Dharmacon (D-011494–03)_siCOPS3 Dharma3: 5’- UCC GAA ACC UGG UGA AUA A), NEDD8 (Dharmacon siGENOME SMARTpool (M-020081–01): 5’-GAA AGG AGA UUG AGA UUG A, 5’-AGA UUG AGA UUG ACA UUG A, 5’-CAG ACA AGG UGG AGC GAA U, 5’-GGA GAU UGA GAU UGA CAU U), and scramble/non-targeting siRNA (Dharmacon D-001206-14-20).

Najor N.A., Fitz G.N., Koetsier J.L., Godsel L.M., Albrecht L.V., Harmon R, & Green K.J. (2017). Epidermal Growth Factor Receptor neddylation is regulated by a desmosomal-COP9 (Constitutive Photomorphogenesis 9) signalosome complex. eLife, 6, e22599.

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