For CD38 deletion in B7-H3.CAR-T cells, 1×106 T cells were transfected with Cas9/RNPs complexes of CD38 targeting guide RNA 5’-AGUGUAUGGGAUGCUUUCAA −3’ (Synthego) using Lonza 4D-Nucleofector™ X Unit (Lonza, AAF-1003X). Transfection was carried out in P3 Primary Cell 4D-Nucleofector™ Solution (Lonza, V4XP-3032) and program E0–115 was used. Immediately after transfection, pre-warmed, incubator equilibrated ImmunoCult media + IL-2 (50 ng/mL) was added to the T cells, followed by culturing for 3 days. CD38 Deletion efficiency was tested 3–6 days post transfection using flow cytometry. Other guides were tested that yielded lower editing efficiency (Supplementary Data Fig. 4b , Supplementary Table 13 .
4d nucleofector x unit
Manufactured by Lonza
Sourced in United States, Switzerland
The 4D-Nucleofector X Unit is a laboratory instrument designed for the delivery of nucleic acids, such as DNA and RNA, into a wide range of cell types. It utilizes proprietary Nucleofection technology to efficiently transfect cells. The device features a compact design and automated functionality to facilitate high-throughput nucleic acid delivery in a controlled and reproducible manner.
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Lab products found in correlation
P1 nucleofection solution, by Lonza (6 mentions)
Pmaxgfp, by Lonza (5 mentions)
P3 primary cell 4d nucleofector x kit, by Lonza (5 mentions)
V4xc 1032, by Lonza (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
P3 primary cell 4d nucleofector x kit l, by Lonza (3 mentions)
Sf nucleofection solution, by Lonza (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
P3 primary cell nucleofector solution, by Lonza (3 mentions)
Grna plasmid, by Addgene (3 mentions)
Dmrc p65 plasmid, by Addgene (3 mentions)
Sf cell line kit, by Lonza (3 mentions)
Nucleocuvette, by Lonza (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Nucleofector solution, by Lonza (2 mentions)
4 oht, by Merck Group (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Chemically modified sgrna, by Synthego (2 mentions)
112 protocols using 4d nucleofector x unit
1
CRISPR-Mediated CD38 Knockout in CAR-T Cells
2
Stable Expression of EGFP-PRIP1 in MCF-7 Cells
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MCF-7 cells stably expressing EGFP-PRIP1 or EGFP-PRIP1(R134Q) were developed previously27 (link). HEK293 cells were transfected with an expression vector encoding EGFP-PLCδPH or a control EGFP vector and cultured in the presence of 1 mg/ml G418 (Nakalai Tesque, Kyoto, Japan) for 14 days. Stably EGFP-expressing colonies were selected. Cells were cultured under conventional growth conditions and transfected with plasmids or siRNA using the Lonza 4D-Nucleofector X Unit (Lonza, Basel, Switzerland) or Lipofectamine 3000 (Invitrogen).
3
Generation of iPSC Lines from PBMCs
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The iPSC line iPSC-UkB-Ctrl-XX was generated from PBMCs via expansion to a cellular intermediate of EPCs, using Stem Span SFEM II (Stem Cell Technologies) and addition of Erythropoietin (R&D Systems), Stem Cell Factor (PeproTech), Interleukin-3 (PeproTech), Insulin Growth Factor-1 (PeproTech), and dexamethasone (Sigma-Aldrich). Following expansion, EPCs were electroporated with Epi5 Reprogramming (Thermo Fisher Scientific) Lonza 4D-Nucleofector X-Unit (Lonza). Cells were plated on Corning ESC-grade Matrigel (Corning) in ReproTeSR (Stem Cell Technologies) before changing to mTeSR 1 (Stem Cell Technologies) when colonies first appeared. Following iPSC clones were picked and expanded before characterization. iPSC-UkB-Ctrl-XX line is cultured on Corning ESC-grade Matrigel and in mTeSR 1. Cells are passaged when colonies are too large using 0.5 mM EDTA in PBS and cultured in a humidified incubator at 37°C and 5% CO2.
4
Electroporation of NCI-H1299 Cells
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Cells were trypsinized and collected, 1e6 cells were resuspended in SF cell line NucleofectorTM solution with 3 μg plasmid DNA. Cells were transferred into 100 μl NucleocuvetteTM Vessel and NCI-H1299 [H1299] cell specific protocol were utilized according to the manufacturer’s protocol (4D-NucleofectorTM X Unit, Lonza). After the pulse application, 100 μL prewarmed D10 medium was added to the electroporated cells in the cuvette. Cells were gently resuspended in the cuvette and transferred into 6 well plate, cultured in incubator. Cells were collected at 24 or 48 hours later for flowcytometry assay and RNA extraction.
5
CRISPR-Cas9 Gene Editing Protocol
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KP1 Cd47 KO and control cells were previously described20 (link). The single guide RNAs (sgRNAs) for CD47, Csf1 and Ccl2 were purchased from Synthego. We added 12 µl of SE buffer (Lonza, V4XC-1032) to each well of a 96-well v-bottom plate. Then 3 µl of sgRNA (300 pmol) was added to the SE buffer. An aliquot of 0.5 µl of Alt-R S.p. Cas9 (Integrated DNA Technologies, 1081059) was added to 10 µl of SE buffer. Next, Cas9 was added to the sgRNA solution, mixed thoroughly and incubated at 37 °C for 15 min to form the ribonucleoproteins. NJH29 cells or KP1 cells were pelleted, counted and resuspended to 106 cells per reaction in 5 µl of SE buffer. Cells and the ribonucleoprotein solution were added to each nucleofection well chamber. Cells were immediately nucleofected using the Lonza 4D-NucleofectorTM X Unit (Lonza, AAF-1002X) with the EN150 program. After nucleofection, warm RPMI medium was added to the cells. The cells were incubated at 37 °C for 15 min and then transferred to a 24-well plate with 1 ml RPMI medium. Editing efficiency was evaluated 4 d later by FACS or RT–qPCR.
6
Investigating LRRK2-mediated Autophagy Regulation
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SH-SY5Y neuroblastoma cell lines stably overexpressing wild-type (WT) or G2019S-LRRK2 were cultured as previously described (herein referred to as WT-LRRK2 and G2019S-LRRK2 cells)33 (link) and were derived as monoclonal cells from a parental culture65 (link). The LRRK2 kinase inhibitor PF-06447475 (herein, PF-475)40 (link) was dissolved in DMSO and applied to cultured cells for 2 h or 6 h, with 0.1% DMSO as vehicle control.
Chloroquine (CQ; 100 µM, 3 h) was used to block to the fusion of autophagosomes with lysosomes and to evaluate the autophagic flux47 (link).
The GFP-LC3-mCherry reporter construct was transfected using FuGene HD (Promega) to analyse the number of autolysosomes. Rab10-RFP constructs were nucleofected using the 4D-NucleofectorTM X unit (Lonza).
Chloroquine (CQ; 100 µM, 3 h) was used to block to the fusion of autophagosomes with lysosomes and to evaluate the autophagic flux47 (link).
The GFP-LC3-mCherry reporter construct was transfected using FuGene HD (Promega) to analyse the number of autolysosomes. Rab10-RFP constructs were nucleofected using the 4D-NucleofectorTM X unit (Lonza).
7
Mfn2 Plasmid Transfection in HeLa Cells
8
Optimizing Electroporation Efficiency for Cell Lines
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Electroporation was performed in 4D-NucleofectorTM X Unit (Lonza # AAF-1002X) device using Lonza NucleocuvetteTM and Ingenio® Electroporation solution. The optimal program was used after finalizing assay conditions and analysing the resultant data sets by flow cytometry (Figure S1 ). Optimization was based around using the Cell line Optimization 4D- NucleofectorTM X Kit (Lonza # V4XC-9064) according to manufacturer protocol to find the optimal solution + pulse condition. Mirus Ingenio electroporation solution with different pulse code was also tested and used. For optimization, cells were spun down (150xg, 10 minutes) and supernatants removed. Cells were resuspended in either Cell Line solution from Lonza with supplements or Ingenio electroporation solution. pmaxGFP from Lonza was added to the cells at an equivalent ratio and cells were electroporated using different pulse codes. Cells were then incubated at RT for 10 minutes, then supplemented with preincubated RPMI media and maintained at 37 °C in 5% CO2 incubator. 48 hours post electroporation, cells were analysed by flow cytometry and the results were calculated as % GFP-positive of gated singlet cells.
9
Restoring USH1C Gene Expression
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For restoring gene expression, 2.5 × 105 primary porcine fibroblasts were transfected with 0.4 µg of an endotoxin‐free pDest_Harm_a1_S/F plasmid (Invitrogen), expressing splice form a1 of the human USH1C gene under the control of the CMV promoter. Transfection was performed with the 4D‐NucleofectorTM X Unit (Lonza), using the P2 Primary Cell 4D‐NucleofectorTM X Kit S and program FS113, according to the manufacturer’s protocol. After transfection, cells were seeded in 10% FCS and analyzed for ciliogenesis as described above.
10
Generation of Ddit3 Knockout BCR-ABL+ B-ALL Cells
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