1.I am using MCF7 and 4T1 cell lines
2. Seeding at 50,000/well concentration in 24 well plate
3. variation among triplicates are high.
4. Sometimes my results differ among control wells, which should not be.
How do you harvest the cells before plating them in the 24 well plate?
Thanks
Extending on the above: Is it possible that you do not have a true single cell suspension? Do you usually check this by microscopy before plating?
Hi,
Confluence is a somewhat undefined unit, and it depends on a few factors. How big are your differences, how do you measure them?
1) Already mentioned, seeding cells as aggregates, usually solid pipetting through ep tip helps. Also keeping cells in suspension as short as possible with constant mixing is a good practice.
2) Counting in dedicated chambers usually generates a noticeable error. There are better methods.
3) It's good to shake plates before resting in thermostat, due to medium viscosity cells tend to group in the middle.
4) Some of the cells could remind unattached (that means more likely dead) because a)developing contamination b)frosting/defrosting cycle c) different "age" or other stressful conditions. Its good to keep your cells for a while before you start your experiment.
If using trypsin/ edta to harvest, please neutralize with serum containing medium.
Thanks to all for your time.
@ Angel A. Rivera,
This is how I harvest my cell before seeding:
1. When i revive cells from -150, I usually go for 3 passages before seeding.
2. When cells are 80-90% confluent, I wash the culture flask 2 times with PBS. Then add 1 ml TrypleE in 25cm3 flask and incubate at 37 degree for 5 minutes. After 5 minutes, I gently pat the flask to detach the cells, add 3 ml of media(C-DMEM) and transfer to 15 ml tubes and centrifuge for 5 min at 120rcf. I dissolve the pellet in 1 ml of media. After counting in a hemocytometer, I take required amount of cell suspension from that 1 ml suspension to make 50 ml of 50,000/ml suspension. From this 50 ml, I add 1 ml in each well of a 24 well plate. I incubate for 24 hours before treatment.
@ Ernst W mullner,
I count the cells in hemocytometer. After seeding check again under microscope.
@ Wojciech Witarski,
I measure cell viability using the following formula:
% Cell viability = (Absorbance of treated sample/ Absorbance of control) × 100
I repeat in triplicates.
Sometimes, I am getting SD 24 for my control wells. Also, sometimes the treated groups show higher cell viability compared to the control, which do not comply with established results.
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