Bone marrow cells were isolated from femur bones as described by Ezeh et al.73 (link). Femurs from each mouse were flushed using a 1 mL syringe and 25-G needle. The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 200 g for 10 min, and resuspended in 20 mL of Isocove’s Modified Dulbeccos Medium ((IMDM) with 2 or 10% FBS, 20 mM L-glutamine, and 100 mg/ml streptomycin and 100 units/mL penicillin). Cell viabilities and concentrations were determined using acridine orange/propidium iodide (AO/PI) staining with a Nexcelom Cellometer Auto 2000 (Nexcelom Bioscience).
Nexcelom cellometer auto 2000
Manufactured by Revvity
Sourced in United States, United Kingdom
The Nexcelom Cellometer Auto 2000 is a cell counter and viability analyzer that uses image-based cytometry technology to measure cell concentration and cell viability in a sample. The device is designed to provide accurate and reliable cell counting and analysis, enabling users to obtain essential data for various research and clinical applications.
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6 protocols using nexcelom cellometer auto 2000
1
Isolation of Mouse Bone Marrow Cells
2
Cytotoxic T cell killing assay
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Cryopreserved polyclonally expanded primary human T cells (frozen on day 7–14 post activation), were thawed on the day of the experiment and counted and assessed for viability using Nexcelom Cellometer Auto 2000 (Nexcelom BioScience, Lawrence, MA, USA). Human T cells and red-fluorescent target cells were then resuspended in Immunocult-XF media supplemented with 100U/mL IL2. Cells were then transferred to a 96-well culture plate at 10 000 T cells per well and 2000 target cells per well. Plates were then placed in an S3 Incucyte Live Cell Imagine system (Sartorius, USA) and incubated at 37°C and 5% CO2 for up to 140 hours. Images were taken every 2 to 4 hours. Target cell growth was tracked using automated assessment of red fluorescent area percentage using the accompanying Incucyte analysis software. Final data was assembled and analyzed using Graphpad Prism.
3
PBMC Isolation and T Cell Activation
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Heparinized whole blood was collected from healthy donors under informed consent by venipuncture and transported at room temperature from Ottawa Hospital Research Institute. Blood was diluted 1:1 with Hank’s balanced salt solution (HBSS) and PBMCs were isolated by Ficoll-Paque™ density gradient centrifugation. Briefly, samples layered on Ficoll-Paque™ gradient were centrifuged for 20 min at 700 × g without applying a brake. The PBMC interface was carefully removed by pipetting and was washed twice with HBSS by stepwise centrifugation for 15 min at 300 × g. PBMCs were resuspended and counted by mixed 1:1 with Cellometer ViaStain™ acridine orange/propidium iodide (AOPI) staining solution and counted using a Nexcelom Cellometer Auto 2000 (Nexcelom BioScience, Lawrence, MA, USA). T cells from were then activated with Miltenyi MACS GMP T cell TransAct™ CD3/CD28 beads and seeded 1x106 T cells/mL in serum-free StemCell Immunocult™-XF media (StemCell Technologies, Vancouver, Canada) with clinical grade 20 U/mL human IL-2 (Novartis, Basel, Switzerland).
4
Isolation and Expansion of Primary T Cells
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To prepare T cells, healthy donor blood samples were obtained under appropriate safety and ethics approvals by the Ottawa Hospital Research Institute (Ottawa, Canada). Whole blood was diluted 1:1 with Hank’s balanced salt solution (HBSS) and PBMCs were isolated by Ficoll-Paque™ density gradient centrifugation, centrifuging for 20 min at 700 × g without applying a brake. The PBMC interface was carefully removed by pipetting and was washed twice with HBSS by stepwise centrifugation for 15 min at 300 × g. PBMCs were resuspended and counted by mixing 1:1 with Cellometer ViaStain™ acridine orange/propidium iodide (AOPI) staining solution and counted using a Nexcelom Cellometer Auto 2000 (Nexcelom BioScience, Lawrence, Massachusetts, USA). T cells from were then activated with Miltenyi MACS GMP T cell TransAct™ CD3/CD28 beads and seeded 1×106 T cells/ml in serum-free StemCell Immunocult™-XF media (Cat#10981, StemCell Technologies, Vancouver, Canada) with 20U/ml clinical grade human IL-2 (Novartis). T cells were then polyclonally expanded for 7 to 10 days in culture before cryopreservation of aliquots using complete media + 10%DMSO.
5
Isolation of Murine Bone Marrow Cells
6
Isolation of Spleen and Thymus Cells
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