Does heavy granulation indicates Mycoplasma contamination in cell lines?

I am looking to incrementally increase calcium ions in culture media to assess the impact on cell proliferation. I was hoping to using CaCl2 anhydrous prills. Can someone advise on how to acheive this? Ie dissolve CaCl2 in dH2O and add to media or dissolve directly into Ca-free DMEM?

How much volume of 100X (Penicillin 10,000 U/mL, streptomycin 10,000 μg/mL) stock should be added to a total 556 ml RPMI medium (500 ml RPMI + 56 ml FBS) to get medium with 1% antibiotic like most frequent way? if the answer was in detail that will be great.

Hello,

I am new to cancer research and I am trying to optimize my small cell lung cancer cell protocol.

I purchased a cell line from ATCC (H1105) and I have been growing the cells in RPMI 1640 media (10% FBS + penicillin/streptomycin.

I am looking for anyone who was worked with these cells in the past and who can let me know if these cells look normal?

I noticed the cells grow very slowly and form spheres.

Am I using the right media? How can I get the cells to grow faster? Can someone refer me to a good SCLC cell culture protocol? Thank you.

Hi, everyone.

first, i'm so sorry about i can't use English well...:(

I research cancer cells and i culture breast cancel cell Sk-br-3.

I use RPMI-1640, 5% FBS, 1% penicillin, streptomycin and incubator: degree 37, 5% CO2

When i see microscope, circles in cells. I think this is different from autophagic vacuoles.

Is that contaminated bacteria or yeast?

I have been trying to grow MRC-5 cells for quite some time. After changing 3 source it still doesn't grow fast enough to do experiment. The cell morphology looks fine visually. I use MEM with 10% FBS, 1% Penicillin Streptomycin, and 1% Non-Essential Amino acids. Change the media each 2-3 days. First I thought its a problem with initial cell inoculation density, so I subsequently tried to put them in smaller surface. With each subculture around 50% cells doesn't survive so the smaller surface area have no effect. Please suggest if you have any solution other than what i mentioned above. Thank you.

I have obtained proteins using PBS containing 18% of penicillin-streptomycin. When I perform BCA the results are extremely high (4.5mg/ml), thus, there is a background of the antibiotics (around 4mg/ml) so I do not know the real concentration of my protein. Do you know any way how I can separate antibiotics from proteins?. Thanks in advance for all your replies. 

our cells obtained from surgical removal tissue .but unfortunately after 24 hours they did not attach to the flusk surface .what is the reason and what should i do? any tips?

1- Wash with PBS 250mg/ml

2-Remove blood vessels and Wash with PBS

3-Crushing and turning the pieces of fat into much smaller and more uniform particles with a scalpel

4-Transfer to Falcon tube and add collagenase 1 at a concentration of 0.075 mg / ml twice the volume of fat and incubate for 60 to 90 minutes in the incubator.

5-After step 4

DMEM culture medium with FBS 10% of the volume inside the Falcon to neutralize collagenase is added to them.

6-Place in a centrifuge for 10 minutes at 2300 RPM

7-The pellet is formed on the wall of the falcon, and after removing the formed phases and remaining the pellet, we add the amount of culture medium into the falcon, and after several pipette pipettes, we transfer the cell into the T-25 flask and give them penicillin. Add 20% Streptomycin and FBS and place in the incubator