rhombus

Two ways to go:-

Try using non tissue culture treated plastics  ..... the cells and beads should not attach to the plastic

If that does not work use a "siliconising fluid" like Repelcote.... this stops cells and beads from attaching to glass and plastic.

Hope this helps

Kindest regards

Uncle Rhombus

Dear CYJ,

From the photo I would agree with Leelee that there does not seem to be any contamination. However if you have a "phase contrast" cell culture microscope, this would give us a better look at both the cell morphology and background media content.

Some cells release alot of debris naturally for that cell line. Debris is easily distinguished from say bacterial contamination when you have experience looking down the micrscope.

Kindest regards

Uncle Rhombus

Dear Vinoth Shanmukam,

"Mammalian cell lysis".........the different methods are:-

Freeze Thaw
Probe Sonication
Ultrasonic water baths (adjustable and constant power)
Homogenisation

Each method is either more or less efficient at cell lysis.

Combined with this is the fact that every mammalian cell is either easier or harder to lyse than the other. So you have these 2 variables to think about. So you have to optimise to get the best returns after lysis.

You should always use a lysis buffer with Protease inhibitors if the resultant protein you want to work with is for westerns or for kinetic studies. The protease inhibitors will increase the chances of having intact active proteins to work with.

There are many kits available for estimating protein concentartion in your lysed cell solution.

Kindest regards

Uncle Rhombus

Serum is just one problem, what about:-

Genetic Drift

Immortalisation

21% Oxygen growing conditions ???????( tissue oxygenation)

Mycoplasma contaminations

Cryptic contaminations

Etc Etc Etc

Kindest regards

Uncle Rhombus

Dear Science noob,

This is an extremely common problem that I have seen over the years. You comment:-

"methods provided in published papers"    .............. this is a big problem in that this part of a paper should probably be the largest section but is commonly just cut to a bear minimum. There are lots of "tricks" that are left out of this section that are essential for thew experiment to be reproduced.

"Same concentration, same cell lines, same conditions"................every cell paper I read states " the cells were grown in a humidified CO2 Incubator at 5% CO2. How many researchers routinely check for humidifiaction AND FYRITE THEIR INCUBATORS TO CHECK PRECISELY THE INTERNAL CO2 CONCENTRATION IN THEIR INCUBATORS.

ARE THE INCUBATORS DIRECT HEAT OR FAN ASSISTED?

WHERE DOE YOUR FOETAL CALF SERUM COME FROM?.......there are massive differences between the qualitof serum sourced for the many countries of the world that produce it.

HOW MANY RESEARCHERS HAVE AUTHENTICATED THEIR CELLS BEFORE USING THEM?

HOW MANY RESEARCHERS ARE WORKING WITH MYCOPLASMA INFECTED CELLS BECAUSE THEY DO NOT TEST THEM?


Does this start to answer your question about reproducibility?

Kindest regards

Uncle Rhombus