The following is adapted from Anderson, et al. (46 (link)). For CRISPR/Cas9 KO of PTPN22, T cells were expanded with Dynabeads and transfected with lentivirus according to the same protocols described above with the following modifications. Virus was removed on day 3 poststimulation instead of day 2, and α-CD3/α-CD28 Dynabeads were removed on day 2 poststimulation instead of on day 5. Dynabeads were removed using an Easy 50 EasySep Magnet (Stemcell Technologies #18002). On day 3, poststimulation T cells were electroporated with 2.5 μg complexed guide RNA (gRNA; Synthego)/Cas9 (Integrated DNA Technologies #260682666) and 20 pmol short, single-stranded oligodeoxynucleotide (ssODN; Integrated DNA Technologies) per 3 × 105 cells. Sequences for the gRNA and ssODN are given below. Samples were electroporated in 20 μL volumes using the P3 Primary Cell 96-well Nucleofector Kit (Lonza #V4SP-3096) and a Nucleofector II + 96-well Nucleofector Shuttle System (Lonza) using program EO-115. Immediately after electroporation, samples were supplemented with 80 μL/well prewarmed hTCM + 100 U/mL IL-2 and incubated 15 min at 37 °C. After incubation, samples were diluted 1:3 in a new 96-well round-bottom plate for a final volume of 250 μL/well hTCM + 100 U/mL. Electroporated samples were maintained in hTCM + 100 U/mL until assays were performed starting on day 11 or day 12.
Easy 50 easysep magnet
Manufactured by STEMCELL
The Easy 50 EasySep Magnet is a laboratory tool used to facilitate the separation of magnetically labeled cells from a cell suspension. It provides a consistent magnetic field to efficiently isolate target cells during the cell separation process.
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Lab products found in correlation
2 protocols using easy 50 easysep magnet
1
CRISPR/Cas9 Knockout of PTPN22 in T Cells
2
Isolation and Lysis of Human Neutrophils
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Neutrophils were isolated from peripheral human blood from healthy donors and Parkinson's patients with VPS35[D620N] mutations as described previously [30 (link)]. For the experimental part of our study using human material, blood was collected with ethical approval for non-clinical research involving human participants granted by the Institutional Review Board of the Medical University of Vienna in Austria, EK Nr: 2195/2016 (for Figure 10 ) or the School Research Ethics Committee, University of Dundee, 19/96 (for Supplementary Figure S12 ). All participants provided informed consent. Viability and cell lysis performed as described previously [30 (link)]. Briefly, Neutrophils were isolated from freshly drawn whole blood from 3 donors (25 ml per donor) using the EasySep Direct Human Neutrophil Isolation Kit and Easy 50 EasySep Magnet (Stemcell) as described [30 (link),31 (link)]. After isolation, neutrophils from each donor were resuspended in RPMI and treated with either the indicated concentration of MLi-2 or DMSO (0.1% v/v). After 1h, cells were harvested in 100 µl per sample of ice-cold lysis buffer supplemented with the potent protease inhibitor diisopropylfluorophosphate (DIFP) to a final concentration of 0.5 mM, that was added to lysis buffer just before lysing cells [31 (link)].
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