Each cell line was trypsinized and counted, and two 25-cm2 culture-treated vented flasks were seeded with 1 × 105 cells each. The media on the cells was changed as described above, and split 1:3 when approaching 90% confluency. One-third of the cells were seeded into a new 25-cm2 flask. Cells were counted on a Z2 COULTER COUNTER Cell and Particle Counter (Beckman Coulter), and the media/cell volume was recorded. The total number of cells was calculated and then multiplied by 3 for each split the line had undergone between the start of the assay and the count to adjust for cells that were removed at each split. If the cells were less than 90% confluent 30 d after the last split, the cells were counted and not cultured further. The data are available in Table S1 .
Z2 coulter counter cell and particle counter
Manufactured by Beckman Coulter
Sourced in United States
The Z2 COULTER COUNTER Cell and Particle Counter is a laboratory instrument designed to measure the size and count of particles, cells, or other suspended objects. It utilizes the Coulter principle to provide accurate and precise measurements.
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Lab products found in correlation
3 protocols using z2 coulter counter cell and particle counter
1
Cell Culture and Counting Protocol
2
CFSE-labeled CD8+ T cell adoptive transfer
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Cells were harvested from the spleen and lymph nodes of donor CD45.1 x OT-I F1 mice, red blood cells lysed. The remaining cells were labeled with 0.625 μM CFSE (Sigma, MA, USA) in PBS for 8 min at 37 °C, then washed and resuspended in PBS. CFSE-labeled cells (4 × 106) were adoptively transferred intravenously (i.v.) into the tail vain of each recipient mouse seven days following lentiviral vector injection. Five days later, cervical lymph nodes were harvested and single cell suspensions were prepared. The total lymphocyte number was determined using a Z2 Coulter Counter Cell and Particle Counter (Beckman Coulter, CA, USA). Cells were washed, incubated with allophycocyanin (APC)-conjugated anti-CD8a mAb (BD Pharmingen, San Jose, CA, USA) and PerCP-conjugated anti-CD45.1 antibody, washed again and resuspended in 0.5 ml FACS buffer (1% BSA and 0.1% sodium azide in PBS). Propidium iodide (PI; 1 μg/ml) was added to samples prior to analysis by flow cytometry.
3
BCL6 Overexpression Impacts Cell Growth
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Cells were cultured to 80% confluence, and then NCI-H1299 cells were transfected with either the BCL6/pcDNA3.1, BCL6ZF/pcDNA3.1 or pcDNA3.1 empty plasmid, by using FuGENE 6 transfection reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. Following a 6-h transfection, 600 µg/ml G418-sulfate (G418; Merck Millipore, Darmstadt, Germany) was added to the medium. Following 2 weeks of G418 treatment, resistant clones were confirmed by RT-qPCR and western blot analysis and selected for further study.
In vitro cell proliferation assay. Cell proliferation was assessed using a Z2 Coulter Counter ® Cell and Particle Counter (Beckman Coulter, Brea, CA, USA). Each cell line (1 ml, 3x10 4 cells) was seeded into 24-well plates. Cell samples were trypsinized and counted with the cell counter, three samples a day, for ~1 week. The numbers were recorded and used to construct growth curves.
Cell cycle analysis. The cells were trypsinized and fixed in 70% ice-cold ethanol for at least 24 h, prior to centrifugation for 5 min at 125 x g, and resuspension in 200 µl PBS. RNase A and propidium iodide were added to a final concentration of 50 µg/ml and 10 mM, respectively, and the cells were incubated on ice for 4 h prior to analysis with a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). A total of 10,000 events were analysed for each sample.
In vitro cell proliferation assay. Cell proliferation was assessed using a Z2 Coulter Counter ® Cell and Particle Counter (Beckman Coulter, Brea, CA, USA). Each cell line (1 ml, 3x10 4 cells) was seeded into 24-well plates. Cell samples were trypsinized and counted with the cell counter, three samples a day, for ~1 week. The numbers were recorded and used to construct growth curves.
Cell cycle analysis. The cells were trypsinized and fixed in 70% ice-cold ethanol for at least 24 h, prior to centrifugation for 5 min at 125 x g, and resuspension in 200 µl PBS. RNase A and propidium iodide were added to a final concentration of 50 µg/ml and 10 mM, respectively, and the cells were incubated on ice for 4 h prior to analysis with a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). A total of 10,000 events were analysed for each sample.
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