Cells as Single-Molecule Protein Proximity Networks
This page collects the public datasets that accompany the manuscript Cells as Single-Molecule Protein Proximity Networks. All sample files are listed under File Download. Two of these experiments are also published as standalone dataset pages, with additional methods detail:
- 1k Human PBMCs (unstimulated and PHA stimulated), proxiome-immuno-155 panel
- CAR-T and Raji Cell Coculture, proxiome-immuno-155 panel with anti-FMC63 spike-in
1k Human PBMCs and Raji cells
Human peripheral blood mononuclear cells (PBMCs) were obtained from buffy coat via density gradient centrifugation using Lymphoprep (Stemcell Technologies). Following isolation, the PBMCs underwent red blood cell lysis and cells were centrifuged three times at 300 x g for 8 minutes to minimize platelet contamination. Subsequently, the cells were fixed with 1% paraformaldehyde for 15 minutes at room temperature.
The same experiment also includes Raji cells (1000 cells). Cells were fixed with 1% PFA for 15 minutes at room temperature as described in UM001: User Manual Proxiome kit Cell Surface v1.00.
Fixed cells were processed as described in UM001: User Manual Proxiome kit Cell Surface v1.00, and sequenced on an Illumina NextSeq2000 sequencing system using 44 cycles for read1 and 78 cycles for read2. Processed with the 155-plex Proxiome panel plus 3 isotype controls. The PXL files on this page are from the manuscript analysis, processed with this panel.
Donor 1 resting and PHA-stimulated PBMCs
PBMCs from one donor were processed as resting cells (1000 cells) or after phytohemagglutinin (PHA) stimulation (500 cells). For PHA stimulation, cells were stimulated with 1.25 µg/ml PHA-L (Invitrogen) for 72 hours, then harvested and fixed with 1% PFA as for resting PBMCs.
Processed with the 155-plex Proxiome panel plus 3 isotype controls.
CAR-T and Raji cell coculture
CD19 CAR-T cells (CD19 scFv-FLAG-TM-CD28-CD3ζ) from PROMAB were evaluated either in isolation or following coculture with CD19⁺ Raji tumor cells from DSMZ (500 cells per sample). For the stimulation conditions, CAR-T cells and Raji cells were mixed at an effector-to-target (E:T) ratio of 1:1 or 5:1. The cocultures were maintained for either 4 hours or 24 hours. Following the incubation period, cells were retrieved and fixed with 1% paraformaldehyde (PFA) for 15 minutes at room temperature. Each sample was processed in duplicate.
Fixed cells were processed according to the DP001: CAR Barcoded Antibody spike-in Proxiome kit (v1.01) manual. Processed with the 155-plex Proxiome panel plus 3 isotype controls, supplemented with an anti-FMC63 probe. Samples were sequenced on an Illumina NextSeq 2000 system using 44 cycles for read 1 and 78 cycles for read 2. The PXL files on this page are from the manuscript analysis, processed with this panel.
Microscopy images
Zip archives of the raw microscopy images that accompany Figure 2, Extended Data Figure 1, and Figure S8 of the manuscript are listed under Microscopy images.
File Download
In order to process the dataset FASTQ files, you need to have the required infrastructure by nf-core/pixelator.
- Download in browser
- Batch download
| Panel file | Size | Checksum (MD5) |
|---|---|---|
| 155-plex Proxiome panel + 3 isotype controls | 6.74 KiB | 183f6a45ae12f74271be1b13c5b43c1c |
| Samplesheet | Size | Checksum (MD5) |
|---|---|---|
| Manuscript analysis samplesheet CSV | 2.65 KiB | db637ca6c17351a86f34e4fd55167b83 |
| 1k Human PBMCs and Raji input files | Size | Checksum (MD5) |
|---|---|---|
| Unstimulated Human PBMCs S02 R1 FASTQ | 10.62 GiB | a01b88348606680d417b8a83aa8d9fb2 |
| Unstimulated Human PBMCs S02 R2 FASTQ | 18.32 GiB | 5591a56624f0dc174fbf91ab7895c616 |
| Raji 1000 cells S06 R1 FASTQ | 8.96 GiB | a3134b2748335abc240eef5b80dc7419 |
| Raji 1000 cells S06 R2 FASTQ | 15.59 GiB | 27cb3b4aa820a92868cff2c1e74fcde4 |
| 1k Human PBMCs and Raji output files | Size | Checksum (MD5) |
|---|---|---|
| Unstimulated Human PBMCs S02 PXL | 3.63 GiB | 94ee29f330a3921460aacb5d5535e4af |
| Raji 1000 cells S06 PXL | 5.59 GiB | a8cb263e90fe8998e62e79759663e52f |
| Donor 1 PBMC input files | Size | Checksum (MD5) |
|---|---|---|
| Donor 1 resting PBMCs S1 R1 FASTQ | 6.74 GiB | 6eba8b954ab5357746f0bc8a8967e10e |
| Donor 1 resting PBMCs S1 R2 FASTQ | 11.04 GiB | 9e09e1c17e23fd625bd8f787d254cd65 |
| Donor 1 PHA-stimulated PBMCs S2 R1 FASTQ | 3.63 GiB | db7ecf013def3755a6fcdc98dd347af1 |
| Donor 1 PHA-stimulated PBMCs S2 R2 FASTQ | 5.97 GiB | 8af23ba58033745477fffb8e5e2d16d3 |
| Donor 1 PBMC output files | Size | Checksum (MD5) |
|---|---|---|
| Donor 1 resting PBMCs S1 PXL | 3.68 GiB | a43605bbac540704ae74a33efaebf853 |
| Donor 1 PHA-stimulated PBMCs S2 PXL | 2.38 GiB | 549a34dee99850514846ba32714fc82b |
| CAR-T coculture output files | Size | Checksum (MD5) |
|---|---|---|
| CAR-T cells replicate 1 S01 PXL | 2.34 GiB | bdda18ffbe3623101f983d149e32e2bd |
| CAR-T cells replicate 1 Raji 4hrs 1to1 S02 PXL | 2.54 GiB | d797bda3666e9453f8e9a30a6b62b6a6 |
| CAR-T cells replicate 1 Raji 4hrs 5to1 S03 PXL | 1.14 GiB | 0c5bea8bd5ed0b132cea72f5c8c554a8 |
| CAR-T cells replicate 1 Raji 24hrs 1to1 S05 PXL | 2.72 GiB | 7acba544b6fd8f98c35aa862cfb7ced2 |
| CAR-T cells replicate 1 Raji 24hrs 5to1 S06 PXL | 2.66 GiB | 15d58ce8943f2fdb10c2610a6693626e |
| CAR-T cells replicate 2 S01 PXL | 2.22 GiB | 4df98fb57ac250c223ad12ca6b9d9919 |
| CAR-T cells replicate 2 Raji 4hrs 1to1 S02 PXL | 2.56 GiB | 7ce8a7515f9da9984d85cba26ac453aa |
| CAR-T cells replicate 2 Raji 4hrs 5to1 S03 PXL | 1.13 GiB | a5a42b74f7a800940ef29f472df52251 |
| CAR-T cells replicate 2 Raji 24hrs 1to1 S05 PXL | 2.59 GiB | 207f544a8fb2c848f0bceb6f76c32251 |
| CAR-T cells replicate 2 Raji 24hrs 5to1 S06 PXL | 2.4 GiB | e929ef7efcc2cebe5caa32519ede4281 |
| Microscopy images | Size | Checksum (MD5) |
|---|---|---|
| Figure 2 microscopy images | 316.7 MiB | f2a1c207da19f91280e755aac44c7174 |
| Extended Data Figure 1 microscopy images | 1.69 GiB | ff4a5d39a15ce295df49861602494a8f |
| Figure S8 microscopy images | 218.42 MiB | 58087a6326575a3c39498027988f070e |
# Panel file
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pna-rnd-158-plex-final.csv
# Samplesheet
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/samplesheet.csv
# 1k Human PBMCs and Raji input files
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA062_unstim_PBMCs_1000cells_S02_S2_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA062_unstim_PBMCs_1000cells_S02_S2_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA062_Raji_1000cells_S06_S6_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA062_Raji_1000cells_S06_S6_R2_001.fastq.gz
# 1k Human PBMCs and Raji output files
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA062_unstim_PBMCs_1000cells_S02_S2.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA062_Raji_1000cells_S06_S6.layout.pxl
# Donor 1 PBMC input files
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA064_Sample_1_S1_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA064_Sample_1_S1_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA064_Sample_2_S2_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA064_Sample_2_S2_R2_001.fastq.gz
# Donor 1 PBMC output files
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA064_Sample_1_S1.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA064_Sample_2_S2.layout.pxl
# CAR-T coculture input files
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_S01_S1_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_S01_S1_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_4hrs_1to1_S02_S2_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_4hrs_1to1_S02_S2_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_4hrs_5to1_S03_S3_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_4hrs_5to1_S03_S3_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_24hrs_1to1_S05_S5_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_24hrs_1to1_S05_S5_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_24hrs_5to1_S03_S6_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_CARTcells_Raji_24hrs_5to1_S03_S6_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_S01_S1_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_S01_S1_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_4hrs_1to1_S02_S2_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_4hrs_1to1_S02_S2_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_4hrs_5to1_S03_S3_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_4hrs_5to1_S03_S3_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_24hrs_1to1_S05_S5_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_24hrs_1to1_S05_S5_R2_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_24hrs_5to1_S06_S6_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_CARTcells_Raji_24hrs_5to1_S06_S6_R2_001.fastq.gz
# CAR-T coculture output files
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA065_CARTcells_S01_S1.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA065_CARTcells_Raji_4hrs_1to1_S02_S2.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA065_CARTcells_Raji_4hrs_5to1_S03_S3.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA065_CARTcells_Raji_24hrs_1to1_S05_S5.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA065_CARTcells_Raji_24hrs_5to1_S03_S6.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA066_CARTcells_S01_S1.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA066_CARTcells_Raji_4hrs_1to1_S02_S2.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA066_CARTcells_Raji_4hrs_5to1_S03_S3.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA066_CARTcells_Raji_24hrs_1to1_S05_S5.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-10a1c79-2025-06-02-dc8eeca6/pixelator/PNA066_CARTcells_Raji_24hrs_5to1_S06_S6.layout.pxl
# Microscopy images
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/images/figure2_microscopy_images.zip
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/images/ED_figure1_microscopy_images.zip
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/images/Figure%20S8%20microscopy%20images.zip
License
Creative Commons Attribution-ShareAlike 4.0 International (CC BY-SA 4.0)
How to Cite
If you are using this data in your research, please cite the Proximity Network Assay pre-print as follows:
Cells as Single-Molecule Protein Proximity Networks. Filip Karlsson, Michele Simonetti, Christina Galonska, Max Karlsson, Hanna van Ooijen, Divya Thiagarajan, Tomasz Kallas, Maud Schweitzer, Ludvig Larsson, Vincent van Hoef, Pouria Tajvar, Johan Dahlberg, Florian De Temmerman, Louise Leijonancker, Vanessa Trombin, Sylvain Geny, Rikard Forlin, Erika Negrini, Beijing Wu, Liu Xi, Stefan Petkov, Lovisa Franzén, Jessica Bunz, Christine Moge, Henrik Everberg, Petter Brodin, Alvaro Martinez Barrio, Simon Fredriksson bioRxiv 2025.06.19.660329; doi: https://doi.org/10.1101/2025.06.19.660329