Cellometer auto t4 cell viability counter

Spleens were removed from euthanized animals under sterile conditions and single cell suspensions of leukocytes were prepared by disaggregation of the tissue through a 100μm nylon mesh (BD Falcon, Bedford, MA). Cells were washed once with RPMI 1640 supplemented with 10% heat-inactivated FBS (hiFBS; Thermo Scientific, Waltham, MA), then incubated with RBC Lysis Buffer (eBioscience, Inc., San Diego, CA) for 1 min to remove red cells, then washed in RPMI 1640+hiFBS. Cells were enumerated at a 1:1 dilution with 0.2% trypan blue stain (Life Technologies, Grand Island, NY) using a Cellometer Auto T4 Cell Viability Counter (Nexcelom, Lawrence, MA), washed, and resuspended at 10⁷ cells/mL in stimulation medium (RPMI 1640 containing 10% FBS [Thermo Scientific, Waltham, MA], 1% sodium pyruvate [Life Technologies, Grand Island, NY], 1% L-glutamine [Thermo Scientific, Waltham, MA], and 0.4% β ME [Sigma-Aldrich, St. Louis, MO]).
Brains and spinal cords were passed through 100μm mesh screens and washed as above. Cells were resuspended in 80 % Percoll (GE Healthcare, Pittsburgh, PA) then overlaid with 40 % Percoll to establish a density gradient and centrifuged at 1,600 rpm for 30min following a method previously described (Campanella et al., 2002 (link)). Leukocytes were collected from the resultant interface, counted, and resuspended in stimulation media for assay.

LNs were removed from euthanized animals under sterile conditions and single cell suspensions of leukocytes were prepared by disaggregation of the tissue through a 100μm nylon mesh (BD Falcon, Bedford, MA). Cells were washed once with RPMI 1640 supplemented with 10% heat-inactivated FBS (hiFBS; Thermo Scientific, Waltham, MA). Cells were enumerated at a 1:1 dilution with 0.2% trypan blue stain (Life Technologies, Grand Island, NY) using a Cellometer Auto T4 Cell Viability Counter (Nexcelom, Lawrence, MA), washed, and resuspended at 10⁷ cells/mL in stimulation medium (RPMI 1640 containing 10% FBS [Thermo Scientific, Waltham, MA], 1% sodium pyruvate [Life Technologies, Grand Island, NY], 1% L-glutamine [Thermo Scientific, Waltham, MA], and 0.4% β-ME [Sigma-Aldrich, St. Louis, MO]).
Spinal cords were passed through 100μm mesh screens and washed as above. Cells were resuspended in 80% Percoll (GE Healthcare, Pittsburgh, PA) then overlaid with 40% Percoll to establish a density gradient and centrifuged at 300 g for 30min following a previously described method(Campanella et al., 2002 (link)). Leukocytes were collected from the resultant interface, counted, and resuspended in stimulation medium for assay.

Immediately prior to transcardial perfusion with Roswell Park Memorial Institute (RPMI, Corning Cellgro, Manassas, VA) 1640, spleens were removed from animals under sterile conditions and single cell suspensions of leukocytes were prepared by disaggregation of the tissue through a 100µm nylon mesh (BD Falcon, Bedford, MA). Cells were washed once with RPMI 1640 supplemented with 10% heat-inactivated FBS (FBS; Thermo Scientific, Waltham, MA), then incubated with RBC Lysis Buffer (eBioscience) for 1 min to remove red cells, and washed in RPMI 1640+FBS. Cells were enumerated in a 1:1 dilution with 0.2% trypan blue stain (Life Technologies, Grand Island, NY) using a Cellometer Auto T4 Cell Viability Counter (Nexcelom, Lawrence, MA), washed, and resuspended at 10⁷ cells/mL in stimulation medium (RPMI 1640) containing 10% FBS, 1% sodium pyruvate (Life Technologies), 1% L-glutamine (Thermo Scientific), and 0.4% β-mercaptoethanol (ME)(Sigma-Aldrich)).
Brains and spinal cords were passed through 100µm mesh screens and washed as above. Cells were resuspended in 80% Percoll (GE Healthcare, Pittsburgh, PA) then overlaid with 40% Percoll to establish a density gradient and centrifuged at 1,600 rpm for 30min following a method previously described (Campanella et al. 2002 ). Leukocytes were collected from the resultant interface, counted, and resuspended in stimulation media for assay.

Spleens were removed from euthanized animals under sterile conditions and single cell suspensions of leukocytes were prepared by disaggregation of the tissue through a 100 μm nylon mesh (BD Falcon, Bedford, MA). Cells were washed once with RPMI 1640 supplemented with 10% heat-inactivated FBS (hiFBS; Thermo Scientific, Waltham, MA), then incubated with RBC Lysis Buffer (eBioscience, Inc., San Diego, CA) for 1 min to remove red cells, then washed in RPMI 1640 + hiFBS. Cells were enumerated at a 1:1 dilution with 0.2% trypan blue stain (Life Technologies, Grand Island, NY) using a Cellometer Auto T4 Cell Viability Counter (Nexcelom, Lawrence, MA), washed, and resuspended at 10⁷ cells/ml in stimulation medium (RPMI 1640 containing 10% FBS [Thermo Scientific, Waltham, MA], 1% sodium pyruvate [Life Technologies, Grand Island, NY], 1% L-glutamine [Thermo Scientific, Waltham, MA], and 0.4% β-ME [Sigma-Aldrich, St. Louis, MO]).
Spinal cords were passed through 100 μm mesh screens and washed as above. Cells were resuspended in 80% Percoll (GE Healthcare, Pittsburgh, PA) then overlaid with 40% Percoll to establish a density gradient and centrifuged at 1600 rpm for 30 min following a previously described method (Campanella et al., 2002 (link)). Leukocytes were collected from the resultant interface, counted, and resuspended in stimulation medium for assay.

Spleens were removed from euthanized animals under sterile conditions and single cell suspensions of leukocytes were prepared by disaggregation of the tissue through a 100µm nylon mesh (BD Falcon, Bedford, MA). Cells were washed once with RPMI 1640 supplemented with 10% heat-inactivated FBS (hiFBS; Thermo Scientific, Waltham, MA), then incubated with RBC Lysis Buffer (eBioscience, Inc., San Diego, CA) for 1 min to remove red cells, then washed in RPMI 1640+hiFBS. Cells were enumerated in a 1:1 dilution with 0.2% trypan blue stain (Life Technologies, Grand Island, NY) using a Cellometer Auto T4 Cell Viability Counter (Nexcelom, Lawrence, MA), washed, and resuspended at 10⁷ cells/mL in stimulation medium (RPMI 1640 containing 10% FBS (Thermo Scientific, Waltham, MA), 1% sodium pyruvate (Life Technologies, Grand Island, NY), 1% L-glutamine [Thermo Scientific, Waltham, MA], and 0.4% β ME (Sigma-Aldrich, St. Louis, MO).
Brains and spinal cords were passed through 100µm mesh screens and washed as above. Cells were resuspended in 80 % Percoll (GE Healthcare, Pittsburgh, PA) then overlaid with 40 % Percoll to establish a density gradient and centrifuged at 1,600 rpm for 30min following a method previously described (Campanella et al., 2002 ). Leukocytes were collected from the resultant interface, counted, and resuspended in stimulation media for assay.