# Search for Entanglement between Spatially Separated Living Systems: Experiment Design, Results, and Lessons Learned

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## Abstract

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## 1. Introduction

## 2. Materials and Methods

#### 2.1. General Structure of CHSH Tests

#### 2.2. Design of the Present Experiment

#### 2.3. Cell Biology Methods

^{®}Live Cell Microtubule-488 dye (Biotium, Fremont, CA, USA), a cell-permeant probe for staining the microtubule cytoskeleton in live cells. PANC-1 cells ($5\times {10}^{3}$) were seeded to 12-well plates the day prior to the treatment day. Before the treatment, a 2X solution of ViaFluo-488 was made by diluting 2 $\mathsf{\mu}$L dye with 1 mL medium followed by addition of 1 $\mathsf{\mu}$L of verapamil to obtain a working solution. The cells were washed with Calcium/Magnesium-free Dulbecco’s PBS, then replaced with ViaFluo-488 working solution and incubated at 37 °C for 30 min. The staining solution was replaced with fresh medium Fluorobrite DMEM for imaging during the treatment. Fluorescence images of PANC-1 cells were taken every minute during the treatment with CytoSmart Lux FL (Axion Biosystems, Atlanta, GA, USA) using Fluorescence Green imaging function. The fluorescence intensity per cell was analyzed by FiJi (ImageJ 2.14.0/1.54f).

^{TM}M7000 Imaging System (Thermo Fisher, Waltham, MA, USA). Fluorescence intensity was quantified by Celleste

^{TM}6.0 Imaging Analysis Software (Invitrogen).

^{TM}Live ReadyProbes

^{TM}Reagent (Invitrogen, Waltham, MA, USA) for 30 min at 37 °C followed by 15 min incubation at room temperature. During the treatment, the green fluorescent and DAPI images of the cells were captured every minute by the EVOS

^{TM}M7000 Imaging System (Thermo Fisher). Fluorescence intensity was quantified by Celleste

^{TM}6.0 Imaging Analysis Software (Invitrogen).

#### 2.4. EEG Methods

#### 2.5. HRV and GSR Methods

#### 2.6. Statistical Methods

- ANOVA over four periods (1 × 4 design): Pre-Baseline, Post-Baseline, Treatment 1, and Treatment 3;
- Paired t-test (1 × 2 design): Treatment 1 and 3 combined versus Pre- and Post-Baselines combined;
- Paired t-test (1 × 2 design): Treatment 1 versus Pre- and Post-periods combined (1 × 2 design);
- Paired t-test (1 × 2 design): Treatment 1 versus Treatment 2;
- Two-way ANOVA (2 × 2 design): Treatment 1 and 3 combined versus Pre- and Post-Baseline combined and cells of type 5 vs. cells of type 4 and 6 combined (cell types 4, 5, and 6 are blinded indices for live cells, cell-free media, and dead cells, in this or a permuted order).

## 3. Results

## 4. Discussion

## 5. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Acknowledgments

## Conflicts of Interest

## Abbreviations

BT | Biofield Therapy |

CHSH | Clauser–Horne–Shimony-Holt |

EEG | Electro-Encephalography |

ER | Einstein–Rosen |

EPR | Einstein–Podolsky–Rosen |

GSR | Galvanic Skin Response |

HRV | Heart Rate Variability |

MEG | Magneto-Encephalography |

NMR | Nuclear Magnetic Resonance |

OR | Objective Reduction |

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**Figure 1.**Expected outcome values for joint measurements of photon pairs at A and B as a function of difference in polarizer angles, assuming perfect classical correlation (red) and entanglement (blue). Optimal angles are indicated by asterisks. From [72], CC-BY license.

**Figure 2.**Biofield therapy (BT) markedly inhibited invasiveness of PANC-1 cells. Invasive potential of PANC-1 cells 48 h after 15 min BT treatment (Treated) or 15 min sham control (Control) for (

**A**) experimental series one and (

**B**) experimental series two. Data are presented as mean ± SD (**** p < 0.0001).

**Figure 3.**Real-time responses of tubulin (

**top**), $\beta $-actin (

**middle**), and ${\mathrm{Ca}}^{2+}$ measures to 15 min BT treatments. Light-gray lines indicate differences between BT-treated PANC-1 cells and sham-treated PANC-1 controls for 40 (tubulin) or 20 ($\beta $-actin and ${\mathrm{Ca}}^{2+}$) individual BT sessions. Orange (tubulin), gray ($\beta $-actin), and blue (${\mathrm{Ca}}^{2+}$) shaded areas indicate 95% confidence intervals around the minute-to-minute average values (orange, gray, and blue lines, respectively). Results for each assay were normalized to the average baseline value, obtained in the one minute prior to initiation of treatment, for that assay and treatment group. Vertical dashed lines indicate the 5 min central period when the BT therapist was allowed to move to relieve discomfort during the treatments.

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**MDPI and ACS Style**

Fields, C.; Cohen, L.; Cusimano, A.; Chakraborty, S.; Nguyen, P.; Deng, D.; Iqbal, S.; Nelson, M.; Wei, D.; Delorme, A.;
et al. Search for Entanglement between Spatially Separated Living Systems: Experiment Design, Results, and Lessons Learned. *Biophysica* **2024**, *4*, 168-181.
https://doi.org/10.3390/biophysica4020012

**AMA Style**

Fields C, Cohen L, Cusimano A, Chakraborty S, Nguyen P, Deng D, Iqbal S, Nelson M, Wei D, Delorme A,
et al. Search for Entanglement between Spatially Separated Living Systems: Experiment Design, Results, and Lessons Learned. *Biophysica*. 2024; 4(2):168-181.
https://doi.org/10.3390/biophysica4020012

**Chicago/Turabian Style**

Fields, Chris, Lorenzo Cohen, Andrew Cusimano, Sharmistha Chakraborty, Phuong Nguyen, Defeng Deng, Shafaqmuhammad Iqbal, Monica Nelson, Daoyan Wei, Arnaud Delorme,
and et al. 2024. "Search for Entanglement between Spatially Separated Living Systems: Experiment Design, Results, and Lessons Learned" *Biophysica* 4, no. 2: 168-181.
https://doi.org/10.3390/biophysica4020012