How to calculate the amount of cell culturing media needed for my fluid-flow cell culturing chamber?

Recently I had my CHO cells cultured in a basal medium (Hycell CHO medium). During the fed batch process, a small amount of high concentration of feed containing Tyr and Cys, which has a high pH (>11), was added into flasks or bioreators. When the two media  got touched together, the cultures became black instantaneous around the adding point, then when the cultures got homogeneous after shaking or stirring,the black would disappear. Now the question is that what reactions have happened? Does it impact on cell growth and how to prevent that? Has anyone experienced this? Thanks!

I am interested to culture (or possibly it is called 'expansion') an human NKT cell line (DN2D5) in vitro for my experiment. Does someone have an 'easy to understand' protocol? Please help. I know these cells need IL-2 (CD3/CD28??), which needs to be removed a couple of days ago before the actual experiment. Please help me with a complete protocol including the composition of culture media.

I am trying to transfect canine Ad-MSCs and NSCs with Bmi-1 plasmid by using lipofectamine 3000, but every time i do the transfected cells start dying after 48hrs, i am using following method for 6-well plate

· Per well

1. 125µL opti MEM + 5µL lipofectamine

2. 125µL opti MEM + 10µg P3000 + 5µg DNA

3. Mix both 1 & 2 and incubation for 20 min then put in cell culture drop by drop

4. after 6 hr serum & antibiotic free culture media is changed to full culture media

the first picture is of cell culture after 24-hrs and 2nd is after 48-hr

Good day, I am having problems with culturing my THP-1 cells. I tried culturing them in several different volumes, I was recommended to seed 5,000,000 cells in 100mL to 150mL and let them sit their for around 1 week to 1 week and a half or so until concentration reaches 1,000,000 cells/mL. Now I have read these cells need cell to cell contact to grow. What I am doing now is seeding 5,000,000 cells in 25mL of RPMI 1640 with a vertical T-75 flask but they still grow slowly and sometimes they are not even viable. Some of my questions regarding to this matter are:

1) Why are my THP-1 cells growing so slowly? I seeded them on a friday at 200,000 cells/mL at 4pm and when I returned on monday at 10am, the medium has barely changed in pH color and I when I observed them through the ZOE microscope, they can be seen most in clusters but there do not appear to be much of them.

2) How do you count these cells or any suspension cells? Every time i try to count THP-1 cells through a hemacytometer, I do not see any cells present. It is very rare to see a cell in the hemacytometer, however, I know my flask has cells because I can see them through the microscope. I tried taking 10uL of cells directly from the flask, I tried taking +/- 100uL of cells and transferring them to an eppendorf tube and from there to count in a ratio of 1:1 and nothing. How exactly do you count suspension cells? Isn't it better to concentrate the cells into a pellet, resuspend the pellet in 1mL of culture media and then count them for a more accurate result rather than taking a small aliquot of culture media directly from the flask and count them?

3) How do you add media to these cells? I have read where people add media every 2 to 3 days to the old media. However, If I add more media to the cells with the pH color being intact and with little cells, aren't I diluting them further? I read these cells need cell to cell contact to grow. When adding even more media on top of the old one, doesn't that increase the passage number? Wouldn't it be better to subculture them to another flask with fresh media?

Hi all,

I was culturing human primary cells with αMEM (prepared from powder according to manufacturer's instruction) on ultra--low attachment binding well plates. The cells were originally from normal culture plates and I dissociated them with Trypsin/EDTA, and put the cell suspension onto the ultra-low binding well plates. According to our hypothesis, these cells are supposed to form spheroids within 1-2 days. The cells clustered together as expected after 2 days, but with the presence of unexpected crystals deposits, both on the bottom of the wells and in the cell clusters.

The medium was prepared with Glutamax as the source of L-Glutamine, and were filtered prior to the addition of FBS. I am pretty sure that I put a big plate of water in the cell culture chamber to make sure the chamber was humidified. What are the possible causes for the crystal formation? And how to get rid of them?

P.S.: The photo below is one of the cell clusters I observed, and you can see clear crystals embedded within the cluster.

We want to dissolve our research samples in cell culture media such as demem or emem. We want to conduct bio-assay guided technique to isolate bio-active compounds. But we have a difficulty to dissolve the samples in the media. We also added 1% of DMSO in the media but the samples were not dissolving. I want an assistant from researchers conducting a similar research.

MTT assay demonstrated that there is no effect of a curcumin derivative at 24 h but shows good effectivity at 48 h. I think this might be due to the slow dispersion of the drug in the cell culture media (Ham's F 12 k). Please help me find a reason with reference.

Hello All, currently i am working to culture sheep kidney and testicle cells and i would like to know the best cell culture media for that

I grown LNCaP cells in an RPMI media with 20% FBS. They are all tested and negative for any mycoplasma. They are being grown in a new incubator. It has been fully filled with water, 11 gallons on Thurs. Feb 20. The level of CO2 is 5% and was calibrated with a Fyrite calibrator on Wed. Mar 4. The temperature is set to 38C under ATCC recommendations and is calibrated with a thermometer that reads the same value. there is a medium sized water basin that is maintained at about half full and check every Friday. The cells present no signs of contamination, with the hood being in its own isolated room, opposite of the entrance into the lab, and is only ever accessed by myself, since I am in a two person lab. I sterilize all materials that enter the hood with 70% EtOH, wear a lab coat and gloves when in the hood room. I am not sure why the cells will not stretch and grow, but instead have decided to have a drop in the population.

Please let me know what I am doing wrong and what I could do to get these cells to grow. They were all growing great and working fine in the old incubator, but due to moving from one lab room to another, the new incubator has just been unable to reproduce the same conditions and are not allowing these cells to grow.