PBMCs were transfected with either GFP control (Lonza) or SOCS3–GFP construct (OriGene) using Amaxa Human T cell Nucleofection kit (Lonza) per the manufacturer’s protocol, with the Amaxa nucleofection program U-014. Cells were stimulated with IL-27 (20 ng/ml; R&D Systems) after allowing cells to rest for 5–7 h after transfection. STAT1 phosphorylation (24 h stimulation) and PD-L1 expression (41 h stimulation) were assessed in live CD3+CD4+ lymphocytes as described above.
Amaxa human t cell nucleofection kit
Manufactured by Lonza
The Amaxa Human T cell Nucleofection kit is a laboratory equipment product that enables the efficient transfection of human T cells. It provides a standardized protocol for the introduction of DNA, RNA, or other molecules into primary human T cells.
Automatically generated - may contain errors
4 protocols using amaxa human t cell nucleofection kit
1
Assessing STAT1 and PD-L1 in IL-27-stimulated PBMCs
2
STAT1 and SOCS3 siRNA Transfection
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PBMCs were transfected with 30 pM STAT1 or SOCS3 siRNA (Thermo Fisher Scientific) using Amaxa Human T cell Nucleofection kit (Lonza) per the manufacturers’ protocols. Control cells were transfected with 30 pM nonspecific, scrambled siRNA. Cells were stimulated with IL-27 (50 ng/ml; R&D Systems) after allowing cells to rest for 5–7 h after transfection. Cells were harvested and stained for pSTAT1 and PD-L1 as per above.
3
SOCS3 Modulation of T-cell Activation
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Naive cells after 48 hours of anti-CD3/CD28 stimulation were transfected with either green fluorescent protein (GFP) control (Lonza, Basel, Switzerland) or 5 mg of SOCS3-GFP construct (OriGene, Rockville, Md) by using the Amaxa Human T-cell Nucleofection Kit (Lonza), according to the manufacturer's protocol, with Amaxa nucleofection program U-014. Cells were cultured under T H 0 or T H 9 conditions for 5 days after transfection. For p-STAT5 analysis, 24 hours after transfection, cells were harvested and stimulated with 80 ng/ mL IL-2 for 30 minutes, and p-STAT5 levels on CD4 1 cells were detected.
4
Silencing STAT1 and TBX21 in T cells
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PBMCs or naive cells after 48 hours of anti-CD3/CD28 were transfected with 30 pmol/L STAT1 or TBX21 small interfering RNA (siRNA; Thermo Fisher Scientific, Waltham, Mass) by using the Amaxa Human T-cell Nucleofection Kit (Lonza), according to the manufacturer's protocol. Control cells were transfected with 30 pmol/L nonspecific scrambled siRNA. Cells were cultured under T H 0 or T H 9 conditions for 5 days after transfection.
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