Hello...
I have worked many years with cells but recently, I have had a doubt!!!... If I have a cellular suspension of 1x106cells/mL and I take 100 uL, I have 1x105 cells in this volume?... Additional, if I seed the 100uL in a well and then I add 100uL of medium or other solvent, the cells will be diluted 1:2?...
That's correct, so your well will contain 1x105 cells at a concentration of 5x105/ mL.
I don't know what your experiment is, or whether your cells are adherent or remain in suspension, but that seems like quite a high plating density for a 96 well plate.
Hi Michael,
I'm working with macrophage J774A.1 (adherence cells)... This cells are for griess assay (nitrite detection), and all the reviews that I have read, use this density... :)
Andres,
As you mentioned, if you have a suspension of 1e6 cells/mL and seed 100 µL of this suspension in a well of a 96-well plate then you will have seeded 1e5 cells in that well (100,000). If you then add 100 µL to that well, you will still have 1e5 cells/well, but it will be 0.5e5 cells/mL. The cells are diluted in the medium, but the absolute number of cells/well remains the same.
J774A.1 are much smaller than the average cell line. For a 96-well plate, 100,000 J774A.1 cells will give you a confluence of 90-95% in the well. This is great for many assays (LDH release, ELISA, other biochemical assays). Seeding 100 µL of a 1e6/mL suspension of J774A.1 is exactly the procedure that I followed for the paper attached below:
Article K+ regulates Ca2+ to drive inflammasome signaling: Dynamic v...
Thanks Jordan!!...
I want to ask if you have had problems with J774A.1 cell culture? My culture, present vacuoles, granules and elongated morphology... I use RPMI+FBS 10% without antibiotics. Do you have any experience?
Vacuoles and granules could be normal, depending on how they present. Can you post a phase or DIC image of your cultures? Elevated vacuolation is a sign of stress and cell activation. You may need to keep the medium more fresh or subculture them more carefully. The ATCC guidelines recommend these cells be grown in DMEM +10% FBS and subcultured by scraping with a sterile cell scraper. If you deviate from these guidelines, you may not be able to maintain normal health of the cells.
J774A.1 should grow with two obvious sub-populations. One is very round, about 12 um diameter. The other is elongated, about 15-25 um in length, give or take. This is normal and healthy. In fact, if you seed cells very low, you will notice after a few days that individual colonies will maintain their round or stretched phenotype, as a whole colony, for a few divisions.
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