P3 primary cell nucleofector kit

Manufactured by Lonza

The P3 primary cell Nucleofector Kit is a laboratory equipment designed for the efficient transfection of primary cells. It provides a platform for the introduction of nucleic acids, such as DNA or RNA, into a variety of primary cell types. The kit includes the necessary reagents and protocols to enable effective transfection, allowing researchers to study gene expression, protein function, and other cellular processes in primary cells.

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Lab products found in correlation

Amaxa nucleofector system, by Lonza (8 mentions) Human il 2, by Thermo Fisher Scientific (4 mentions) Soluble anti cd28 cd28.2, by BD (4 mentions) Anti cd3 cd3 2, by Mabtech (4 mentions) Dual luciferase reporter assay system, by Promega (3 mentions) Smartapool c fos sirna, by Horizon Discovery (2 mentions) Smartapool pdcd4 sirna, by Horizon Discovery (2 mentions) Prl sv40 renilla luciferase reporter, by Promega (2 mentions) Ap 1 luciferase reporter plasmid, by Addgene (2 mentions) Nucleofection, by Lonza (1 mentions) Amaxa 4d nucleofector, by Lonza (1 mentions) Accutase, by Merck Group (1 mentions) Amaxa 4d nucleofector system, by Lonza (1 mentions) Cm 150, by Lonza (1 mentions) Y 27632, by Selleck Chemicals (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Tgf β1, by Thermo Fisher Scientific (1 mentions) Anti cd3 anti cd28 beads, by Thermo Fisher Scientific (1 mentions) Sd208, by Selleck Chemicals (1 mentions) 4d nucleofector system, by Lonza (1 mentions)

Spelling variants of this product

Nucleofector kit (131 citations) Kit P3 (2 citations)

12 protocols using p3 primary cell nucleofector kit

Silencing miR-21, c-FOS, and PDCD4 in T cells

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To silence miR-21, naive CD4⁺ T cells were transfected with either locked nucleic acid (LNA) miR-21 inhibitor or scrambled inhibitor negative control (Exiqon) using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). After 48 hours, PDCD4, PTEN, SPRY1 and b-actin expression was assessed by western blot. To silence c-FOS or PDCD4 expression in activated cells, naive CD4⁺ T cells activated for 36 hours were washed and transfected with either SMARTApool c-FOS siRNA, SMARTApool PDCD4 siRNA or negative control siRNA (Dharmacon) using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). Cells were resting for 12 hours and cultured on plates coated with 1 mg/ml anti-CD3 (CD3–2; Mabtech) plus 2 ¼g/ml soluble anti-CD28 (CD28.2; BD Biosciences) and 10 U/ml human IL-2 (Peprotech). After 3 days, cells were harvested and analyzed.

Kim C., Hu B., Jadhav R.R., Jin J., Zhang H., Cavanagh M.M., Akondy R.S., Ahmed R., Weyand C.M, & Goronzy J.J. (2018). Activation of miR-21-Regulated Pathways in Immune Aging Selects against Signatures Characteristic of Memory T Cells. Cell reports, 25(8), 2148-2162.e5.

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Silencing miR-21, c-FOS, and PDCD4 in T cells

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Optimized Nucleofection for Gene Editing

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1 × 10⁶ K562 cells were transfected with 2 μg TALEN-encoding plasmid and 5 μg donor plasmid (unless otherwise indicated) by nucleofection (Lonza) using program T-016 and a nucleofection buffer containing 100mM KH2PO4, 15mM NaHCO3, 12mM MgCl2 • 6H20, 8mM ATP, 2mM glucose, pH 7.4. 4 × 10⁵ CD34+ HSPCs were nucleofected with an Amaxa 4D Nucleofector with the P3 Primary Cell Nucleofector Kit (V4XP-3032) and program EO-100 per the manufacturer’s instructions. 1 × 10⁶ H1 cells were transfected with 0.5 μg or 2.5 μg of each TALEN-encoding plasmid and 4 μg donor plasmid (unless otherwise indicated) by nucleofection (Lonza) using an Amaxa 4D Nucleofector (program B-105) with the P3 Primary Cell Nucleofector Kit (V4XP-3032) and following manufacturer’s instructions.

Hendel A., Kildebeck E.J., Fine E.J., Clark J., Punjya N., Sebastiano V., Bao G, & Porteus M.H. (2014). Quantifying Genome Editing Outcomes at Endogenous Loci using SMRT Sequencing. Cell reports, 7(1), 293-305.

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Genetic Modulation of Activated CD4+ T Cells

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Naïve CD4⁺ T cells were transfected with either SMARTpool negative control siRNA, SMARTpool TFEB siRNA, SMARTpool MYC siRNA or SMARTpool VPS39 siRNA (all from Dharmacon) using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). Cells were rested for 2 hours, washed and activated by dynabeads for 5 days. Cells were then harvested and analyzed.

Jin J., Kim C., Xia Q., Gould T.M., Cao W., Zhang H., Li X., Weiskopf D., Grifoni A., Sette A., Weyand C.M, & Goronzy J.J. (2021). Activation of mTORC1 at late endosomes misdirects T cell fate decision in older individuals. Science immunology, 6(60), eabg0791.

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T Cell Activation and Transcriptional Assay

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Naive CD4⁺ T cells were activated with anti-CD3/anti-CD28 beads and transduced with a lentiviral vector as described above. After 36 hours, activated cells were cotransfected with the AP-1 luciferase reporter plasmid (Plasmid #40342; Addgene) along with pRL-SV40 renilla luciferase reporter (Promega) using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). After 4 hours, activated cells were cultured on plates coated with 1 μg/ml anti-CD3 (CD3–2; Mabtech) plus 2 mg/ml soluble anti-CD28 (CD28.2; BD Biosciences) and 10 U/ml human IL-2 (Peprotech). On day 3 after activation, cells were lysed and luciferase reporter activity was measured with the Dual-Luciferase Reporter Assay System (Promega).

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Efficient Gene Editing of Human iPSCs

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Human iPSCs were enzymatically dissociated with Accutase (Sigma) and plated on Matrigel coated dishes at 1:3 ratio in E8 supplemented with 10 um Y-27632 (Selleck Chemicals). 24 – 48 hr later, human iPSCs were dissociated with Accutase into single cells. ~2×10⁶ cells were transfected with a pair of TALENs (1.0 μg of each TALEN) by nucleofection using the Amaxa 4D Nucleofector system (Lonza) with the P3 Primary Cell Nucleofector Kit and program CM-150 per manufacturer’s instructions (Lonza). Following nucleofection, iPSCs were re-suspended in 1 ml pre-warmed E8 supplemented with 5 μM Thiazovivin and then plated in 6-well plates pre-coated with Matrigel and allowed to recover for 48 hr.

Karakikes I., Termglinchan V., Cepeda D.A., Lee J., Diecke S., Hendel A., Itzhaki I., Ameen M., Shrestha R., Wu H., Ma N., Shao N.Y., Seeger T., Woo N., Wilson K.D., Matsa E., Porteus M.H., Sebastiano V, & Wu J.C. (2017). A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases. Circulation research, 120(10), 1561-1571.

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Luciferase Assay for IL9 Promoter

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The human IL9 promoter region from −406 to +361 bp was cloned into a pGL3‐basic plasmid. pGL3 basic plasmids or pGL3‐IL9, the Renilla luciferase reporter pR‐TK, and the indicated genes were co‐transfected into HEK 293T cells using FuGENE HD Transfection Reagent (Promega). Forty‐eight hours after transfection, luciferase activity was determined using the dual luciferase reporter assay system (Promega). Alternatively, pGL3‐IL9 and the Renilla luciferase reporter pR‐TK were transfected into naïve CD4 T cells on day 4 after activation using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). Three days after transfection, luciferase activity was determined.

Hu B., Li G., Ye Z., Gustafson C.E., Tian L., Weyand C.M, & Goronzy J.J. (2019). Transcription factor networks in aged naïve CD4 T cells bias lineage differentiation. Aging Cell, 18(4), e12957.

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Modulating FOXP3 Exon Splicing

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Enhanced FOXP3 exon splicing was achieved using Fluorescein-labelled Morpholino Antisense Oligonucleotides (MAO) with the following sequences: 5′-TGCCCATTCACCGTCCATACCTGGT-3′ for FOXP3ex1/3, 5′-AGCTGTGAAATGGCACAAACATGAG-3′ for FOXP3ex6/8 and 5′-CCTCTTACCTCAGTTACAATTTATA-3′ as a control (GeneTools). Prior to activation, T cells were transfected with 15 μM MAO using the P3 Primary Cell Nucleofector Kit and a Nucleotransfector device (Lonza) according to the manufacturer’s instructions.

Mailer R.K., Joly A.L., Liu S., Elias S., Tegner J, & Andersson J. (2015). IL-1β promotes Th17 differentiation by inducing alternative splicing of FOXP3. Scientific Reports, 5, 14674.

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T cell activation and autophagy regulation

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Freshly isolated human naïve CD4⁺ T cells or naïve CD8⁺ T cells were transfected with either SMARTpool negative control small interfering RNA (siRNA), SMARTpool TCF7 siRNA, SMARTpool CISH siRNA, SMARTpool ATP6V1A siRNA (all from Dharmacon), or pCMV6-Entry Mammalian or Human CISH expression vector (NM_145071, both from Origene) using the Amaxa Nucleofector system and the P3 primary cell Nucleofector Kit (Lonza). After transfection, cells were rested for 2 hours before being activated by anti-CD3/anti-CD28 Dynabeads for 3 or 5 days. For lentiviral transduction, naïve CD4⁺ T cells were transduced with a lentiviral vector expressing either scrambled control, TCF7, RFP-LC3 or RFP-GFP-LC3 (as the autophagic flux probe). After overnight culture, cells were activated by anti-CD3/anti-CD28 Dynabeads for 3 days.

Murdoch D.R., Laing R.T., Mills G.D., Karalus N.C., Town G.I., Mirrett S, & Reller L.B. (2001). Evaluation of a Rapid Immunochromatographic Test for Detection of Streptococcus pneumoniae Antigen in Urine Samples from Adults with Community-Acquired Pneumonia. Journal of Clinical Microbiology, 39(10), 3495-3498.

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Differentiation of Naïve T Helper Cells

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Isolated naïve CD4 T cells were activated with anti‐CD3/anti‐CD28 beads (Life Technologies) at a 1:5 ratio and cultured for 7 days under the following conditions: TH1, 10 ng/ml IL12 and 1 μg/ml neutralizing anti‐IL4 antibody (Ab); TH2, 20 ng/ml IL4 and 10 μg/ml neutralizing anti‐IFNγ Ab; and TH9, 10 ng/ml IL4 and 5 ng/ml TGFβ1. To generate Tregs, naïve CD4 T cells were cultured on plates coated with anti‐CD3 (5 μg/ml) and anti‐CD28 antibody (1 μg/ml) in the presence of IL2 (2 ng/ml) and TGFβ1 (5 ng/ml). All cytokines were purchased from PeproTech. All antibodies were from R&D Systems. TGFβ receptor inhibitor SD‐208 was purchased from Selleck Chemicals.
siRNA for TGFβR3, BATF, IRF4, and plasmids expressing BCL6 or ID3 were transfected into naïve CD4 T cells on day 3 after activation using the Amaxa Nucleofector system and P3 primary cell Nucleofector Kit (Lonza). TF expressions were examined on day 5 and the frequencies of cytokine‐producing cells were determined on day 7 after restimulation. siRNA were purchased from Dharmacon Inc. and plasmids were from Genscript.

Hu B., Li G., Ye Z., Gustafson C.E., Tian L., Weyand C.M, & Goronzy J.J. (2019). Transcription factor networks in aged naïve CD4 T cells bias lineage differentiation. Aging Cell, 18(4), e12957.

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