1.Validation on adherent cell lines from different species and tissue types

Human cell lines (HEK293, HeLa, SH-SY5Y, LX-2* and IMR-90), mouse cell lines (NIH/3T3 and C2C12), and the hamster cell line CHO were plated in 96-well culture plates at different cell densities and incubated at 37°C, 5% CO2 for 24 hours. Cells were then lysed with 50 µL of supplemented lysis buffer #4 for 30 minutes at RT under gentle shaking, and 4 µL of cell lysate (neat or prediluted in the supplemented lysis buffer) were transferred into a low volume white microplate. For HTRF detection, 12 µL of kit diluent were added followed by the dispensing of 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

 

 

 

 

 

 

 

 

 

The HTRF Alpha-Tubulin Housekeeping kit is adapted to adherent cells from different tissue types (kidney, brain, liver, lung, muscle, ovary, cervix…). It has been validated on samples from human, mouse and hamster origin, and is also compatible with rat, monkey, dog and pig species. The assay range and sensitivity have been optimized on the most commonly used cell lines, using classical cell densities to avoid cell lysate predilution before HTRF detection. For just a few cell lines that express high levels of alpha-tubulin (e.g. C2C12 and LX-2*), the lysate must be prediluted in the supplemented lysis buffer before transfer to the detection plate (dilution factor between 1/2 and 1/10 at the most).

2. Validation on suspension cell lines and primary cells

The human lymphoblast cell lines Jurkat and TK6, as well as PBMCs isolated from human blood samples, were plated in half 96-well plates at different cell densities (under 30 µL) and directly lysed with 10 µL of supplemented lysis buffer #4 (4X) for 30 minutes at RT under gentle shaking. For HTRF detection, 4 µL of neat cell lysate were transferred into a low volume white microplate and 12 µL of kit diluent were added before the dispensing of 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. The assay is also suitable for working on common cell densities of T- and -B lymphoblast cell lines and PBMCs, without the need to predilute cell lysates.

 

 

3. Validation on tissue samples

Liver tissue lysates from three DIN (Diet-Induced NASH) mice* were prepared with lysis buffer #3 and analyzed as described in the technical note “Optimize your HTRF® cell signaling assays on tissues”. After total protein quantitation, samples were adjusted to the same concentration and serially diluted in the supplemented lysis buffer. For HTRF detection, 4 µL of each tissue lysate dilution were transferred into a low volume white microplate and 12 µL of kit diluent were added, before the dispensing of 4 µL of premixed detection antibodies. The HTRF signal was recorded after an overnight incubation at RT. The Alpha-Tubulin Housekeeping assay is compatible with tissue lysates, with an assay range adapted to samples containing low, medium and high concentrations of proteins. *DIN mouse liver samples were kindly provided by the preclinical CRO Physiogenex (Labège, France).

 

 

 

4. Correlation with the BCA protein assay

Lysates of human and mouse cell lines plated at different cell densities in 96-well culture plates for 24 hours were prepared as described in Section 1. Serial dilutions of liver tissue lysates from DIN (Diet-Induced NASH) and MCD (Methionine- and -Choline Deficient) mice* were prepared as described in the previous section. For HTRF detection of the housekeeping protein alpha-tubulin, 4 µL of cell or tissue lysate were transferred into a low volume white microplate before the addition of 12 µL of kit diluent and 4 µL of premixed detection antibodies. In parallel, the concentration of proteins was determined in the same lysates using the BCA protein assay (QuantiPro™ BCA Assay Kit, Sigma-Aldrich) according to the manufacturer’s intructions. For both methods (HTRF or BCA), lysates were analyzed either neat or prediluted in the appropriate buffer in order to work within the linear range of each kit. The cell densities used for the correlations are mentioned on each graph next to each point.