Dear Gabriela De Larrañaga

Please have a look at the protocol in our publication:

Article MYSM1-dependent checkpoints in B cell lineage differentiatio...

Hopefully that helps with your experiments.

All the best & good luck,

Michael

Thank Michael.

I can't see the Alamar Blue test

Hi Gabriela De Larrañaga

Sorry I attached the wrong B cell paper. You find the protocol here and the results in Figure 2.

Article Regulation of B Lymphocyte Development by Histone H2A Deubiq...

If I remember correctly, we did 1/10 of total volume of the culture medium for the Alamar Blue concentration. For details please see the materials and methods.

I hope that helps.

All the best & kind regards,

Michael

Dear Gabriela De Larrañaga

Alamar Blue contains resazurin and additional compounds to prevent the over-reduction of resazurin to a non-fluorescent product. These additives also slow the rate of generation of the fluorescent product. Consequently, alamar blue assay will require longer incubation times compared to resazurin.

Alamar blue reagent may be simply added as 10% of the sample volume (i.e., add 10 µL alamar blue reagent to 100 µL sample), followed by a 1–4 hour incubation at 37ºC. But you need to know that the optimum incubation time may vary between cell types. Since you did not observe any color change even after 4 hours of incubation, longer incubation time may be used for greater sensitivity.

However, if alamar blue is used for extended incubation periods, there is a possibility that the buffering agents present in alamar blue will reach their maximum buffering efficiency. At this point, the equilibrium between the oxidized and reduced forms of alamar blue is disrupted, and a colorless form of the dye may be obtained. This could result in an apparent bleaching of the solution, and a decrease in the amount of the reduced (red) form of alamar blue. So, I would recommend that you perform pilot experiments to optimize incubation time for alamar blue. The link below may help you with these experiments.

https://www.bio-rad-antibodies.com/determine-optimum-cell-density-incubation-time-for-a549-p388-cells-alamarBlue-spectrophotometry.html

Please note: If you are using resazurin for the assay, then you may dissolve resazurin in DPBS (pH 7.4) to 0.15 mg/ml. Filter-sterilize the resazurin solution through a 0.2 μm filter into a sterile, light protected container and use 20 µl resazurin solution to each well containing 100 µl/well of final solution.

Best.

Thank you Michael so much !!!!

Thank you so much, Malcolm. Yes, I read all about bio-rad presentations. Perhaps I need more time for incubation.