Murine T cells were isolated from splenocytes as previously described (Pegram et al., 2012 (link)). Murine CAR T cells were maintained in culture with RPMI in the presence of 100 IU/mL recombinant human IL-2 (Proleukin; Novartis, Basel, Switzerland), selected with Nylon Wool Fiber (Polysciences, Warrington, PA), and activatedwith CD3/CD28 beads, accordingto manufacturer’s instructions (Gibco, Thermo Fisher, Waltham, MA). Human T cells were derived from fresh blood-derived leukocyte concentrate (Leukopack) obtained from the New York Blood Center. Mononuclear cells were separated using density gradient centrifugation with Accu-prep (axis-Shield PoC AS, Oslo, Norway). T cells were isolated, activated, and expanded with 2 × 106/mL PHA (Sigma Aldrich, St. Louis, MO). T cells were cultured in RPMI 1640 in the presence of 100 IU/mL recombinant human IL-2 (Proleukin). Viable cells were enumerated using flow cytometry and counting beads (Ebioscience), following manufacturer’s protocol. Activated human and mouseT cells were retrovirallytransduced as previously described (Pegram et al., 2012 (link), 2015 (link)). CAR expression was detected using Armenian hamster 12D11 antibody (anti-CD19 CAR) or an Alexa Fluor 647-conjugated hamster antibody that specifically binds the 4H1128z CAR (Monoclonal Antibody Facility, Memorial Sloan Kettering Cancer Center).
Nylon wool fiber
Manufactured by Polysciences
Sourced in Switzerland
Nylon Wool Fiber is a synthetic fiber composed of nylon. It is a versatile material commonly used in various textile applications.
Automatically generated - may contain errors
Lab products found in correlation
Counting beads, by Thermo Fisher Scientific (2 mentions)
Cd3 cd28 beads, by Thermo Fisher Scientific (2 mentions)
Accu prep, by Axis-Shield (2 mentions)
Phytohemagglutinin (pha), by Merck Group (2 mentions)
Recombinant human il 2, by Novartis (2 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
3 protocols using nylon wool fiber
1
Murine and Human T Cell Isolation and Activation
2
Murine and Human T Cell Isolation and Activation
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Murine T cells were isolated from splenocytes as previously described (Pegram et al., 2012 (link)). Murine CAR T cells were maintained in culture with RPMI in the presence of 100 IU/mL recombinant human IL-2 (Proleukin; Novartis, Basel, Switzerland), selected with Nylon Wool Fiber (Polysciences, Warrington, PA), and activatedwith CD3/CD28 beads, accordingto manufacturer’s instructions (Gibco, Thermo Fisher, Waltham, MA). Human T cells were derived from fresh blood-derived leukocyte concentrate (Leukopack) obtained from the New York Blood Center. Mononuclear cells were separated using density gradient centrifugation with Accu-prep (axis-Shield PoC AS, Oslo, Norway). T cells were isolated, activated, and expanded with 2 × 106/mL PHA (Sigma Aldrich, St. Louis, MO). T cells were cultured in RPMI 1640 in the presence of 100 IU/mL recombinant human IL-2 (Proleukin). Viable cells were enumerated using flow cytometry and counting beads (Ebioscience), following manufacturer’s protocol. Activated human and mouseT cells were retrovirallytransduced as previously described (Pegram et al., 2012 (link), 2015 (link)). CAR expression was detected using Armenian hamster 12D11 antibody (anti-CD19 CAR) or an Alexa Fluor 647-conjugated hamster antibody that specifically binds the 4H1128z CAR (Monoclonal Antibody Facility, Memorial Sloan Kettering Cancer Center).
3
Isolation of Human T Cells from PBMCs
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Human PBMCs were purified from the whole blood of healthy human donors (Taipei Blood Bank, Taiwan). RBCs were removed by ACK lysis buffer (Gibco BRL, CA), and the remaining cells were incubated in a culture dish for 30 min at 37°C to remove attached cells. Unbound cells were panned again in a culture dish precoated with goat anti-human Ig for 30 min at 37°C to remove B cells. T cells were loaded in a column packed with nylon wool fiber (Polyscience, Inc.) for 1 h at 37°C in a 5% CO2 humidified incubator, and T cells were collected from the column and used immediately.
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