Spleens were harvested, mashed over a 40-µm cell strainer (VWR, 10199 – 654 ), and CD8+ T-cells purified by positive magnetic bead selection (Miltenyi Biotec, 130-117-044 ) according to manufacturer’s instructions. Purified CD8+ T-cells were counted and cell diameter measured using a Moxi Z mini automated counter (Orflo, MXZ001 ). The cells were characterized using Flow cytometry described below or plated at 1 × 106 (24-well plate) or 5 × 105 (48-well plate) and activated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher, 11456D ) in a 1:1 beat to T-cell ratio and cultured in 2 ml (24WP) or 1 ml (48WP) RPMI 1640 (Thermo Fisher, 21875 ) supplemented with 10% Fetal Bovine Serum, (Thermo Fisher, 10270 – 106 ), 50 µM 2-mercaptoethanol (Thermo Fisher, 21985023 ), 10 U/ml penicillin-streptomycin (Thermo Fisher, 15140122 ), and 10 U/ml recombinant human IL-2 (Sigma, 11011456001 ), and incubated at 37 °C for 3 days in a humidified CO2 incubator.
40 m cell strainer
Manufactured by Avantor
Sourced in United States
The 40-µm cell strainer is a laboratory equipment used to filter and isolate cells or particles based on size. It features a 40-micron mesh that separates materials smaller than 40 microns while retaining larger particles.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Magnetic bead positive selection, by Miltenyi Biotec (2 mentions)
Recombinant human il 2, by Merck Group (2 mentions)
Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Complete rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Carl Roth (1 mentions)
B cell isolation kit, by Miltenyi Biotec (1 mentions)
2 mercaptoethanol, by Carl Roth (1 mentions)
3 protocols using 40 m cell strainer
1
Isolation and Activation of Mouse CD8+ T Cells
2
Purification and Activation of Murine CD8+ T Cells
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Spleens were harvested from 8-12 week old C57BL/6J (RRID : IMSR_JAX:000664) mice, mashed over a 40 µm cell strainer (VWR, 10199-654), and CD8+ T cells were purified by positive magnetic bead selection (Miltenyi Biotec, 130-117-044) according to manufacturer’s instructions. Purified CD8+ T cells were counted and cell diameter measured using a Moxi Z mini (Orflo, MXZ001) or a TC20 (Bio-Rad, 145-0101) automated counter. 1x106 (24-well plate) or 5x105 (48-well plate) CD8+ T cells were activated with Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher, 11456D) in a 1:1 bead to T cell ratio and cultured in 2 ml (24-well plate) or 1 ml (48-well plate) RPMI 1640 (Thermo Fisher, 21875) supplemented with 10% Fetal Bovine Serum (Thermo Fisher, 10270-106), 50 µM 2-mercaptoethanol (Thermo Fisher, 21985023), 100 U/ml penicillin-streptomycin (Thermo Fisher, 15140122), and 10 U/ml recombinant human IL-2 (Sigma, 11011456001), and incubated at 37˚ C for 3 days in a humidified CO2 incubator.
3
Isolation and Culture of Murine B Cells
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Spleens were removed from naïve C57Bl/6j mice (8-12 weeks). Single cell suspensions of splenocytes were obtained by dispersion through a 40 µM cell strainer (VWR, Radnor, Pennsylvania, USA). B cells were isolated by negative selection using the B Cell Isolation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany). The procedure was carried out following the instructions of the manufacturer. Purified B cells were cultured in 24 well plates (TPP, Trasadingen, Switzerland) at 10 6 cells/well in 1 ml RPMI 1640 complete medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10 % heat-inactivated fetal calf serum (FCS, Thermo Fisher Scientific, Waltham, Massachusetts, USA), 2 mM L-glutamine (Roth, Karlsruhe, Germany) and 50 µM 2-mercaptoethanol (Roth, Karlsruhe, Germany) and kept at 37 °C and 5 % CO2.
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