High Content Screening (HCS) Reagent Kits From Cellomics

We tested five of the Cellomics HCS Reagent Kits on our primary retinal cell culture to try to find the light-induced cell death pathway of photoreceptors that have been exposed to light. We tested ERK MAPK, NFкβ, p38 MAPK, STAT 3, P53 P21, and NFкβ and c-Jun. The results from each of the kits were mixed. It appears that most of the pathways tested were not activated and/or difficult to interpret without the Cellomics ArrayScan® HCS Reader.

In most, if not all, of the kits, activation is determined when the target molecule, which is often phosphorylated, moves from the cytoplasm to the nucleus. At this point, the cell death pathway has been activated. Therefore, an activated cell will have the target co-located over the Hoechst/DAPI stain, or nucleus. The stain, in theory, will get brighter because of the condensing of the nucleus prior to cell death. Because of this mechanism of activation, the ArrayScan® Reader can determine co-location of the stained target inside the nucleus much easier than you can by viewing it under the microscope. I took DAPI images and over-laid the targets and even then, it was very difficult to tell which cells were being activated.

At this point, I will diverge and go into the details of one of the kits: NFкβ and c-Jun, which seemed to work in our system. I will compare their protocol to ours. In their protocol, the permeabilization step is done followed by rinsing with blocking buffer, which I assume is 5% goat serum. Then the antibodies are added to the blocking solution and incubated at room temperature for 1 hour. To me, there doesn’t seem to be anything magical in their solutions. My guess is that our standard protocol would work just as well. We use a slightly harsher fix protocol where we add ice cold methanol after fixing with formaldehyde. I’ve heard that a methanol fix might destroy very sensitive targets. I followed their protocol, used their reagents, and used a one-step formaldehyde fix. Our protocol permeabilizes, blocks, and stains for 1 hour at 37ºC. I didn’t bother to test our protocol side by side with theirs, but I could not help to be curious. My gut instinct tells me our standard cytochemical antibody (ICC) protocol would work as well as theirs did.

Therefore, I would recommend you buy one kit and test it versus your standard ICC protocol. If your protocol works as well or better than theirs then I would recommend you save your money and just buy the antibodies separately from a reliable source. It seems these kits are overpriced, and as far as I can tell, you can prepare your own regents for a fraction of the cost Cellomics will charge you for their secret sauces.