Can someone help me out with the details. I am trying to create a routine calculation for hemocytometer and volume dilutions for my experiments. Here is what I have. Culture HepG2 in EMEM=10%FCS to 80% confluence on T75. Trypsinize (5ml) , add equal volume (5ml complete media) into falcon tube and spin 1000g for 5 minutes. Aspirate supernatant and re-suspend in 1ml of complete media. Now Count: taking 100ul of cells + 400ul trypan OR 100uL +100 uL trypan (which do you guys prefer?). Count 4 large squares. Calculate avg live cells (today was 404) and avg dead (today 20). calculate percent viability (got it). calculate cell density [avg live x dil factor (2 or 5 based on above) x 10000]. for today that gave me 20,525,000 cells/ml. Now I have to convert this #/ml into 10, 50 or 100k cells/ well on 96well flat bottom plate with a total vol of 100uL per well (any best practices here?). Sooooooo how do I make the jump statistically? I know I am going to need a total volume of 10ml per plate so how to create a simple method (maybe in excel?) do do this calculation routine. I would like to scale this up once I get good at understand the dilutions. Since I am doing toxicity assay (dose response curves) I am not sure what min/max number of cells should be per well. I will be using MTT as an endpoint for 24 and 48 plates to start.
Hello
For Cell Seeding
Add 50μL (for 96-well plate) or 300μL (for 24-well plate) of culture medium in each well ( In case solution contains bubbles when medium placing, they should be removed by pipetting or centrifugation less than 400G).
100μL/well (for 96-well plate; Cell density: 3.3x105 viable cells/mL) or 500μL/well (for 24-well plate; Cell density: 4x105 viable cells/mL) of cell suspension was added in each well.
The plates were gently shaken in a back-and-forth and side-to-side manner to disperse cells evenly in each well. Avoid cells’ clumping in the center of wells.
The plates were carefully placed into a water-jacketed incubator under 5% CO2 and 95% air at 37℃.
And see this link :
https://www.researchgate.net/post/How_to_achieve_a_uniform_monolayer_with_HepG2_cells_seeded_in_24_well_plate_format
Good Luck
For seeding HepG2 cells in 96-well plate genrally uses 1x104 - 5x105 (I use 5x104, but it's depends of the cell), with 100ul final volume.
For the 24-well plate you can use 5x105 - 1x106.
Hi Antonio.
I am about to start a very similar experiment using HepG2 and MTT for a cytotoxicity assay.
I would be very grateful if you wouldn't mind sharing the protocol you found that worked for you?
Thank you very much!
Best wishes,
Lucy
Thanks everyone for threw great responses. So essentially I settled on 50000 cells per well, at 100 microliters total volume per well. And this instance therefore I would need roughly 500,000 cells per ml to start. Download the Hemotap app, plug in your numbers and it will spit out the volumes you would need to achieve your desired seeding density. for me basically that means growing cells in a T25 flask Tupac 8% confluency then detaching and spinning down Andre suspending in 1 ml complete media or 5ml complete media. Recently I've been doing it in 5 ml. from the there, i use hemotap to get me to 500,000 cells per ml, because that number essentially means that when I pipet 100microliters of that solution to my 96 Well, each well will have 50,000cells in it. Also important to note that for the exp im running right now I don't need the whole 96-well titer plate so I don't need 10 ml of seed stock.
Hello, Antonio,
HepG2 cells tend to grow in clusters, so in our lab we usually do not plate them so that they reach a confluent monolayer. In this article, we have visually and qualitatively described the relationship between cell seeding density and confluence after 24 h in 96-well plates: https://www.academia.edu/40969549
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