For the cryomedium, why do you need 90% FBS? Usually when I freeze mamallian cells, I use regular medium that is 10% FBS and add 10% DMSO to it. Is there something critical about suspension cells that requires 90% FBS?
During centrifugation, the centrifugal force (RCF) acts on particles in suspension, causing them to sediment based on their size and density. However, rotor design plays a crucial role in how this force is applied to the cells and impacts their behavior. Fixed-Angle Rotors vs Swing-out Rotors • Uneven Sedimentation: In fixed-angle rotors, the sample tubes are held at a fixed angle relative to the rotational axis. This creates a sedimentation gradient within the tube. Cells closer to the bottom experience a higher RCF compared to those near the top of the tube. In the case of swing-out rotors, the sample tubes swing outwards. It allows for a more uniform distribution of the RCF throughout the sample volume. • Shear Stress: The uneven distribution of RCF can generate shear stress on the cells, particularly those sedimenting at the bottom. This shear stress can damage fragile cells like HEK293, impacting cell viability and integrity. Whereas, swing-out rotors minimize shear stress on the cells. This gentler centrifugation helps preserve the viability and integrity of fragile cells like HEK293, making them ideal for downstream applications.
Thanks for the video. how did you get the dilution factor 4 after mixing 10 ul of cells in 10 ul of trypan blue during counting? I though it should be 2, am I wrong?
+fefe adding 10 uL cells into 10 uL of trypan blue makes a dilution factor of 2. Then repeat it to the second trypan blue drop makes another dilution factor of 2. 2X2 = 4
I am facing problems in growing Kasumi-6 cell line. I'm using RPMI 1640 with 20% FBS and then directly do the flask I'm adding 2ng/ml GM-CSF, l-glutamine and antibiotics. But they not growing I don’t know where is the problem.
The appropriate volume of freezing media is determined by considering two key factors: the desired final cell concentration post-thaw and the potential for cell loss during the freezing process. Typically, we aim to fill cryogenic vials to 70-80% capacity with freezing media containing a slight volume excess to account for this loss. This approach ensures sufficient viable cells are available for successful experimentation after thawing.
Normally you follow manufacturing instructions about the ratio for subculturing. They will say 1:4 to 1:6 or something like that. Therefore, you won't need to count the cells.
You can estimate cell number by visual inspection and use a standard split ratio (like 1:2 or 1:3). Monitor cell health closely and adjust as needed. Regular cell counting is recommended for accuracy.
For the cryomedium, why do you need 90% FBS? Usually when I freeze mamallian cells, I use regular medium that is 10% FBS and add 10% DMSO to it. Is there something critical about suspension cells that requires 90% FBS?
Thank you for an educative video. Do have any video that shows cell stimulation of whole blood?
Yes i have some information regarding this.
Can fixed-angle rotors be used to centrifuge HEK293 cells instead of swing-out rotors?
During centrifugation, the centrifugal force (RCF) acts on particles in suspension, causing them to sediment based on their size and density. However, rotor design plays a crucial role in how this force is applied to the cells and impacts their behavior.
Fixed-Angle Rotors vs Swing-out Rotors
• Uneven Sedimentation: In fixed-angle rotors, the sample tubes are held at a fixed angle relative to the rotational axis. This creates a sedimentation gradient within the tube. Cells closer to the bottom experience a higher RCF compared to those near the top of the tube. In the case of swing-out rotors, the sample tubes swing outwards. It allows for a more uniform distribution of the RCF throughout the sample volume.
• Shear Stress: The uneven distribution of RCF can generate shear stress on the cells, particularly those sedimenting at the bottom. This shear stress can damage fragile cells like HEK293, impacting cell viability and integrity. Whereas, swing-out rotors minimize shear stress on the cells. This gentler centrifugation helps preserve the viability and integrity of fragile cells like HEK293, making them ideal for downstream applications.
Thanks for the video. how did you get the dilution factor 4 after mixing 10 ul of cells in 10 ul of trypan blue during counting? I though it should be 2, am I wrong?
+fefe adding 10 uL cells into 10 uL of trypan blue makes a dilution factor of 2. Then repeat it to the second trypan blue drop makes another dilution factor of 2.
2X2 = 4
I am facing problems in growing Kasumi-6 cell line. I'm using RPMI 1640 with 20% FBS and then directly do the flask I'm adding 2ng/ml GM-CSF, l-glutamine and antibiotics. But they not growing I don’t know where is the problem.
How do you determine the appropriate volume of freezing media?
The appropriate volume of freezing media is determined by considering two key factors: the desired final cell concentration post-thaw and the potential for cell loss during the freezing process. Typically, we aim to fill cryogenic vials to 70-80% capacity with freezing media containing a slight volume excess to account for this loss. This approach ensures sufficient viable cells are available for successful experimentation after thawing.
During the subculture u don't take cell count so how we clear take how much cell in ml and how much take growth medium for subculturing.
Normally you follow manufacturing instructions about the ratio for subculturing. They will say 1:4 to 1:6 or something like that. Therefore, you won't need to count the cells.
You can estimate cell number by visual inspection and use a standard split ratio (like 1:2 or 1:3). Monitor cell health closely and adjust as needed. Regular cell counting is recommended for accuracy.
Our company manufactures measuring cylinder of hexagonal base.
Good Video! Thank you.
What song/Instrumental is this?
Thank you it’s very well done, good quality
「ビデオコンテンツはとても素晴らしいです、おめで
Does suspension cellline gets cloudy after 1 day
No, cloudiness is usually an indicator of contamination.
Thank you
SONG PLS?
Thank you ;)
Thank you for an educative video. Do have any video that shows cell stimulation of whole blood?