The short guide RNA was designed using the online tool provided by CHOPCHOP (http://chopchop.cbu.uib.no )36 (link). Freshly isolated naïve CD4 T cells were activated with plate bound anti-CD3 and CD28 antibodies. Cas9 proteins were prepared immediately before experiments by incubating 1 μg Cas9 with 0.3 μg sgRNA in transfection buffer at room temperature for 10 min. 24 h after T cell activation, these cells were electroporated with a Neon transfection kit and device (Thermo).
Neon transfection kit and device
Manufactured by Thermo Fisher Scientific
The Neon transfection kit and device is a laboratory equipment designed to facilitate the introduction of nucleic acids, such as DNA or RNA, into cells. The kit includes the Neon transfection device and the necessary reagents for the transfection process. The device utilizes an electroporation-based method to temporarily create pores in the cell membrane, allowing the nucleic acids to enter the cells.
Automatically generated - may contain errors
Lab products found in correlation
Neon kit, by Thermo Fisher Scientific (3 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Neon electroporation device, by Thermo Fisher Scientific (2 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Thermo Fisher Scientific (1 mentions)
Gapdh sirna, by Thermo Fisher Scientific (1 mentions)
Il 15, by Thermo Fisher Scientific (1 mentions)
Egfp mrna, by TriLink (1 mentions)
8 protocols using neon transfection kit and device
1
CRISPR-Cas9 Editing of Activated CD4 T Cells
2
CRISPR-Cas9 Activation of Memory Th2 Cells
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Four short-guide RNAs were designed using the online tool provided by CHOPCHOP (http://chopchop.cbu.uib.no ) or E-CRISP (http://www.e-crisp.org/E-CRISP/ ). Oligonucleotides pairs with BbsI-compatible overhangs were annealed and cloned into the expression vector pGEM-T Easy-T3-BB-sgRNA for in vitro transcription. Memory Th2 cells (bulk targeted cell cultures) were activated with plate-bounded anti-TCRβ and CD28 antibodies. Cas9 proteins were prepared immediately before experiments by incubating 1 μg Cas9 with 0.3 μg sgRNA indicated below in transfection buffer at room temperature for 10 min. Twenty-four hours after T cell activation, these cells were electroporated with a Neon transfection kit and device (Thermo). And then, these cells were cultivated with IL-7, IL-25, and IL-33 for 4 days for further expansion.
sgDusp10: 5’- CTTGAGGGTCACAACGGCGG-3’
sgControl (luciferase): 5’-CGTATTACTGATATTGGTGGG-3’
sgDusp10: 5’- CTTGAGGGTCACAACGGCGG-3’
sgControl (luciferase): 5’-CGTATTACTGATATTGGTGGG-3’
3
Transfection and gene expression analysis
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Cultured ILC2s were electroporated with a NEON transfection kit and device (Thermo Fisher Scientific) using three pulses of 1800 V for 10 ms to transfect select siRNAs or pCMV-myc-JunB vector. After 24 h of transfection, cells were collected and used for RT–qPCR or RNA-seq. Primary hepatocytes were transfected with RNAi using Lipofectamine RNAi Max (Thermo Fisher, 13778075) according to the manufacturer’s instructions. The siRNAs were as follows: Ct si (Negative Control #1, Cat# AM4635) and Target si (Cat# 4390771, 4390815). The IDs were as follows: Gata3 si: s61810, Junb si: s68567, Pa2g4 si: s201806, Calr si: s63270, Jund si: s201553, Runx1 si: s201126, Runx3 si: s63447, Il4ra si: s68281, Il13ra1 si: s68211, Stat3 si: s74452, Stat6 si: s74463. The pCMV-myc-JunB vectors used were described previously44 (link).
4
CRISPR-Cas9 Editing of Sptlc1 and Sptlc2 in Activated T Cells
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The short guide RNA was designed using the online tool provided by CHOPCHOP 29 (link). Freshly isolated naïve CD4 T cells were activated with plate bound anti-CD3 and CD28 antibodies. Cas9 proteins were prepared immediately before experiments by incubating 1 μg Cas9 with 0.3 μg sgRNA in transfection buffer at room temperature for 10 min. 24 h after T cell activation, these cells were electroporated with a Neon transfection kit and device (Thermo).
Target sequences used in this study are shown below.
sgSptlc1 : 5′- TTTGTCGTAGAATCCTCGCAAGG-3′
sgSptlc2 : 5′- GCGGAACATTGGTGTAGTTGTGG-3′
Target sequences used in this study are shown below.
sgSptlc1 : 5′- TTTGTCGTAGAATCCTCGCAAGG-3′
sgSptlc2 : 5′- GCGGAACATTGGTGTAGTTGTGG-3′
5
Ceacam-1 siRNA Transfection in PBMCs
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Seven frozen PBMC samples were electroporated with a Neon transfection kit and device (Invitrogen). Cells (3 × 105) were incubated for 18 h with 10 ng/ml of IL-2 (Peprotech) and 10 ng/ml of IL-15 (Peprotech) and resuspended in 10 µl of buffer T (Neon kit, Invitrogen). Ceacam-1 siRNA (Entrez Gene ID 634; detected transcripts NM_001024912.2, NM_001205344.1, and NM_001712.4) or negative control siRNA (at a final concentration of 100 nM; Ambion) were added to the cell suspension. Ten microliters of the suspension were electroporated (1,700 V, 20 ms, three pulses). GAPDH siRNA (Ambion) was used as a positive control to evaluate efficiency of the silencing. Cells were incubated for 24 h at 37°C and 5% CO2 and then stimulated in anti-CD16-coated plate (1 µg/ml) for 5 h at 37°C and 5% CO2 in the presence of CD107a antibody. Brefeldin A (eBioscience) and Monensin (eBioscience) were added during the last 4 h of incubation. Surface markers and functions by intracellular staining were assessed by flow cytometry as described earlier.
6
Antisense Oligonucleotide Transfection of T Cells
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Antisense 2′-o-methyl oligonucleotides (IDT) were transfected using a Neon transfection kit and device (Invitrogen). A total of 5 × 105 T cells that have been stimulated for 1 day were washed two times with PBS before suspension in 10 μL of buffer T (Neon kit, Invitrogen). Antisense oligos (1 μL of 125 μM) were added to the cell suspension to a final volume of 11 μL and mixed. 10 μL of the suspension was electroporated with a Neon electroporation device (Invitrogen; 1,600 V, 10 ms, three pulses). The transfected T cells were then cultured in 0.5 mL media so that the final concentration of tRF antisense oligos is 250 nM. The 0.5 mL media for culturing transfected T cells were in 24-well plates with plate-bound anti-CD3 (2.0 μg/ml), soluble anti-CD28 (0.5 μg/ml) and 10 units/mL IL2. After 2 days of culture, the transfected T cells were harvested and analyzed by flow cytometry for surface CD44 and CD62L expression. The sequence of antisense oligo for Leu-TAA:5′tRF was CCACUCGGCCAUUCUG, for Leu-TAG:5′tRF was CCGCUCGGCCACGCUA, for Ser-GCT:3′i-tRF was CCCUCGCGUGCAAAGCACA, and for Leu-TAA:3′i-tRF was ACGCGGAUAUAAAUCC.
7
CRISPR-mediated Editing of KEAP1 Gene
8
Antisense Oligonucleotide Transfection of T Cells
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