By Jun Seita, M.D., Ph.D.
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine
Detection of a GFP signal driven by the Oct3/4 promoter was extensively used to define STAP cells in the original STAP papers [1, 2] and similar approaches have been employed by many third-party attempts to reproduce STAP cells. On the other hand, it is well known that stressed and/or damaged cells harbor significant auto-fluorescence. This is particularly pertinent to the case of STAP cells, because the various stresses applied to cells may themselves induce cellular autofluorescence independent of Oct3/4-GFP reporter activation. Neither flow cytometry nor a fluorescent microscope can distinguish the origin of an observed fluorescent signal, e.g. GFP. Rather such equipment only measures the intensity of a photon within a certain range of wavelengths, as determined by light source and filters used. Distinguishing an authentic Oct3/4-GFP signal from auto-fluorescence is the critical first step to reproduce STAP cells. Thus, just following the original and updated protocol  provided by the authors is insufficient to lend confidence to the claim that low-pH treatment converts terminally differentiated cells into Oct3/4-GFP expressing cells.
As a stem cell biologist who totally relies on flow-cytometry technology, I’d like to propose one major addition to the protocol.
Please process cells isolated from wild-type mice in parallel with cells from Oct3/4-GFP transgenic mice all the way through the experiment and analyze them exactly side-by-side.
All other factors should be identically matched, namely background strain; age; gender of mice; low-pH treatment; and the subsequent culture process. This is the only negative control that can definitely distinguish an authentic Oct3/4-GFP signal from auto-fluorescence. This strategy named Fluorescence-minus-one (FMO) control is the most reliable way to define the boundary between positive and negative cells in modern flow-cytometry . Non-stress-treated but cultured cells are not apropos for this purpose because the spectrum and intensity of auto-fluorescence varies based on the condition of the cells. Moreover, this is an addition, not a modification of the protocol, and thus the body of the original protocol remains intact.
The flow-cytometry data presented in Extended Data Figure 5g of the Nature letter  suggest that some controls relating to flow-cytometry might need to be revisited throughout the STAP studies and it might be worth determining other factors that need to be matched. Each sample should be analyzed using the exact same acquisition parameters on the flow-cytometer. The voltage setting of the photomultiplier tubes (which define acquisition sensitivity) and compensation parameters should be optimized and fixed before commencing analysis, and should not be adjusted between samples. Also the authors should declare what fluorochrome/fluorescent protein was used in each figure. Referring to the protocols [1-3] provided for what antibodies were employed in the original studies, there are 29 options for anti-CD90 from eBioscience, 22 options for anti-CD19 from Abcam, 4 options for anti-CD34 from Abcam, and 6 options for anti-Integrin alpha 7 from R&D Systems. The ambiguity over what exact antibodies were precisely used makes it challenging to reproduce the authors’ exact procedures. Also since many fluorochromes/fluorescent proteins have emission spectra that are wider than the filters for a particular detector, one always needs to deal with spillover of fluorescence into adjacent detectors by a method known as “compensation”. Perfectly compensating for spillover for certain complex combinations of distinct fluorescent emissions can be quite challenging, e.g. simultaneous analysis of GFP, PE and auto-fluorescence. Thus it is very important to know what combination of fluorochromes/fluorescent proteins were used to interpret the presented flow-cytometry plot in the figure. If one is not familiar with voltage setting, compensation, choice of combination of fluorochromes, or FMO, it is highly recommended to consult with flow-cytometry experts in your flow-cytometry core facility.
The addition of these simple, yet critically important steps and controls will produce far clearer data.
Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, Yamato M, Vacanti CA.
Nature. 2014 Jan 30;505(7485):641-7. doi: 10.1038/nature12968.
Obokata H, Sasai Y, Niwa H, Kadota M, Andrabi M, Takata N, Tokoro M, Terashita Y, Yonemura S, Vacanti CA, Wakayama T.
Nature. 2014 Jan 30;505(7485):676-80. doi: 10.1038/nature12969.
3. Essential technical tips for STAP cell conversion culture from somatic cells
Haruko Obokata, Yoshiki Sasai & Hitoshi Niwa
Protocol Exchange (2014) doi:10.1038/protex.2014.008
Published online 5 March 2014
Tung JW, Heydari K, Tirouvanziam R, Sahaf B, Parks DR, Herzenberg LA, Herzenberg LA.
Clin Lab Med. 2007 Sep;27(3):453-68, v. Review.