Commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) collected from healthy human donors (ePBMCs; Cellular Technology Limited) were thawed and washed with ice cold Miltenyi MACS Buffer. NK cells were enriched from PBMCs using the Miltenyi NK Cell Isolation Kit (Miltenyi #130-092-657) following the manufacturer’s instructions. Isolated NK cells were counted and adjusted to a density of 1E6 cells/mL in assay media (RPMI-1640 media+ 5% ultra-low IgG FBS [Gibco #A3381901 + rhIL-2 100 U/mL [R&D Systems # 202-IL]). The primary NK cells (1E5 cells/well) were cultured in a 96-well V-bottom cell culture plate overnight at 37°C, 5% CO2 prior to use in stimulation assays.
Epbmc
Manufactured by Cellular Technology
Sourced in United States
EPBMCs (Enriched Peripheral Blood Mononuclear Cells) are a type of lab equipment used for the isolation and enrichment of specific immune cells from blood samples. The core function of EPBMCs is to separate and concentrate peripheral blood mononuclear cells, which include lymphocytes and monocytes, for further analysis or experimental purposes.
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Lab products found in correlation
Miltenyi nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Macs buffer, by Miltenyi Biotec (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Cell trace red, by Thermo Fisher Scientific (1 mentions)
Automacs separator, by Miltenyi Biotec (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Lm511 e8 matrix, by Nippi (1 mentions)
Stemfit medium, by Ajinomoto (1 mentions)
6 protocols using epbmc
1
Isolation and Culture of Primary Human NK Cells
2
Integration-free iPSC-derived NSPC protocols
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Three lines of integration free human PBMC-derived iPSCs (1210B2, 1231A3, and 1201C1), which were established from ePBMCs® from the Cellular Technology Limited (OH, USA) at Center for iPS Cell Research and Application (CiRA: Kyoto, Japan) by an integration-free method [51 (link)], were used. The 1210B2 and 1231A3 iPSCs were cultured with a feeder-free protocol [33 (link)], and the 1201C1 iPSCs were cultured with an on-feeder protocol that uses SNL feeder cells. They were induced into NSPCs as previously described [52 (link), 53 (link)] with two slight modifications. Briefly, in the first protocol, the NSPCs were induced directly from embryoid bodies (EBs) by a protocol that consists only of a floating culture. In the second protocol, the EBs were adhered to laminin-coated culture dishes on day 7, and they subsequently formed neural rosettes (NRs), which were picked on day 14. We refer to NSPCs induced directly from EBs as EB-NSPCs, and those induced from the NR phase as NR-NSPCs. The NSPCs were expanded using the neurosphere culture technique [23 (link), 54 (link)].
3
Induced Neuronal Progenitor Cells from iPSCs
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Human induced pluripotent stem cells (1210B2), which were established from peripheral blood mononuclear cells (ePBMCs®, Cellular Technology Limited, OH, USA) at the Center for iPS Cell Research and Application, Kyoto University (CiRA: Kyoto, Japan) by an integration-free reprogramming method, were induced into neural progenitor cells using the dual SMAD-inhibition method with dorsomorphin plus SB431542 as previously described [65 (link), 66 (link)]. hiPSCs were differentiated into stage 3 neurons [64 (link), 67 (link)], then fixed with 4% PFA and 0.1% glutaraldehyde in 0.1 M PBS for 15 min at room temperature, and immunostained as described previously [68 (link)]. For visualization of F-actin, cells were incubated with rhodamine-phalloidin (1:500; Molecular Probes, Eugene, OR). Images were obtained by a CCD Camera (CoolSNAP HQ2, Photometrics).
4
Treg-mediated Suppression of T Cell Proliferation
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Treg cells were isolated from recipient PBMCs and commercial normal PBMCs (ePBMC®, Cellular Technology Limited, Shaker Heights, Cleveland, USA; as a healthy control) using the CD4+CD25+CD127dim/− Treg Isolation Kit α (Miltenyi Biotec, Bergisch Gladbach, Germany) and an AutoMACS separator (Miltenyi Biotec). Isolated Treg cells labelled with CellTraceRed (CTR, Invitrogen) were co‐cultured with autologous sorted CD8+CD25− and CD4+CD25− T cells (as responder cells) labelled with CellTraceViolet (CTV, Invitrogen) in the presence of anti‐CD3/CD28. A total of 2 × 104 responder T cells were co‐cultured at different ratios with isolated autologous Treg cells. After 5 days of co‐culture, the proliferative activity of the CD8+ responder T cells was measured by calculating the percentage of dividing CTVlo cells. The percentage of suppression was calculated as [% Suppression = 100 − {(division index of responder T cells only)/(division index of responder T cells in co‐culture with Treg cells)} × 100].
5
Generation of SNCA-Deficient hiPSCs from 1231A3 Line
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The 1231A3 hiPSC line derived from ePBMC®, purchased from Cellular Technology Limited (http://www.immunospot.com/ ), it was established by Kyoto University, and provided by the RIKEN Bioresource Research Center (Tsukuba, Japan) through the National BioResource Project of the Ministry of Education, Culture, Sports, Science, and Technology/Japan Agency for Medical Research and Development (MEXT/AMED), Japan [20 (link)]. Herein, we used SNCA−/− hiPSCs generated from the 1231A3 hiPSC line [21 (link)]. The hiPSC-related experiments were approved by the Ethical Review Committee for Medical and Health Research Involving Human Subjects, Kyoto Pharmaceutical University.
6
Retinal Tissue Differentiation from Human iPSCs
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Human iPSC-1231A3 line was established by Kyoto University, derived from ePBMC (Cellular Technology Limited; http://www.immunospot.com/ ) [21 (link)]. Human iPSCs were maintained on LM511-E8 matrix (Nippi) in StemFit medium (Ajinomoto) according to the published protocol [21 (link)]. The differentiation to retinal tissue was conducted following the culture methods designated SFEBq, BMP and Induction-reversal culture described previously [10 (link)] with minor modification at the initial phase in differentiation (Kuwahara et al., in submission). Human iPSC-retinas at differentiation day (DD) 58–78 were cut into small pieces of approximately 0.5 × 2 to 1 × 1.5 mm2 for transplantation.
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