To investigate the proliferative capacity of mouse MAIT cells in vivo, we isolated naïve B6-MAITCAST LMNCs and non-parenchymal hepatic mononuclear cells (HMNCs), which were immediately pooled and subjected to magnetic MAIT cell sorting. Isolated MAIT cells were then expanded ex vivo following a recently published protocol by Parrot et al. [43 (link)]. MAIT cell-depleted fraction was irradiated at 35 Gy and used as a source of feeder cells, which were seeded at 2 × 105 cells/well along with 2 × 104 MAIT cells/well of a U-bottom plate. Cells were maintained in ImmunoCult T Cell Expansion Medium (STEMCELL Technologies) supplemented with 20 ng/mL of recombinant mouse interleukin (IL)-2 (R&D systems), 8% CTS Immune Cell Serum Replacement (Thermo Fisher), 100 μg/mL of Normocin (InvivoGen), and 100 U/mL Penicillin/Streptomycin. Cultures were split twice a week and replenished with fresh medium and feeder cells. After 3 weeks, expanded MAIT cells were washed, labeled with 1 μM CellTrace Far Red dye (Thermo Fisher), and injected i.v. at 5 × 105 cells/B6-MAITCAST mouse. Twenty-four hours after adoptive transfer, the recipients were given PBS or immunized with PR8 and 5-OP-RU i.p. Three days later, mice were sacrificed for several organs in which MAIT cell proliferation was assessed by flow cytometry.
Immunocult t cell expansion medium
Manufactured by STEMCELL
ImmunoCult T Cell Expansion Medium is a serum-free, animal component-free cell culture medium designed for the in vitro expansion of human T cells. It provides the necessary nutrients and growth factors to support the proliferation of T cells while maintaining their functional properties.
Automatically generated - may contain errors
Lab products found in correlation
Cts immune cell serum replacement, by Thermo Fisher Scientific (2 mentions)
Celltrace far red dye, by Thermo Fisher Scientific (1 mentions)
Normocin, by InvivoGen (1 mentions)
Nk92 cells, by Thermo Fisher Scientific (1 mentions)
Countbright beads, by Thermo Fisher Scientific (1 mentions)
Alpha mem, by Corning (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Nk 92 cells, by American Type Culture Collection (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
2 protocols using immunocult t cell expansion medium
1
Expanding and Tracking Mouse MAIT Cells
2
Characterization of CAR T-cell Interactions with NK-92 Cells
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NK-92 cells (ATCC) were cultured in minimum essential medium alpha (MEM alpha, Corning) supplemented with 12.5% heat-inactivated FBS (Corning), 12.5% heat-inactivated horse serum (Sigma-Aldrich), 1X l -glutamine (GlutaMAX, Gibco), 1X antibiotic–antimycotic (Gibco), and 5 ng/mL rhIL-2 (Peprotech). Cell seeding was performed at a density of 3×105 cells/mL, and the culture medium was refreshed every 2 to 3 days. NK-92 cultures were maintained for a maximum of three weeks, with a total of 6–9 passages. NK-92 cells were tested for Mycoplasma contamination. CAR T cells were thawed and rested for 72 hours in ImmunoCult T-cell expansion medium (STEMCELL Technologies) supplemented with 5% CTS immune cell serum replacement (Gibco) and 100IU rhIL-2 (PeproTech). CAR T cells were labeled with CTV (Thermo Fisher Scientific) and incubated with NK-92 cells at a 1:1 E:T ratio for ≤72 hours. At each time point, coculture samples were analyzed via flow cytometry for expression of CD56, HLA-E, and HLA-ABC and labeling of CTV. Information about the antibodies used can be found in Supplementary Table S4). CountBright Beads (Invitrogen) were added to coculture samples before acquisition and used to calculate cell counts. Counts of HLA-E+ and HLA-ABC+ CAR T cells were compared and normalized with a 0 hours time point.
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