CAR-T and Raji Cell Coculture, proxiome-immuno-155 panel with anti-FMC63 spike-in
Introduction / Cell preparation
CD19 CAR-T cells (CD19 scFv-FLAG-TM-CD28-CD3ζ) from PROMAB and CD19⁺ Raji tumor cells were evaluated either in isolation or following coculture with CD19⁺ Raji tumor cells from DSMZ. 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 to capture dynamic surface remodeling and intercellular interactions. 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 to ensure technical reproducibility.
PNA workflow
Fixed cells were processed according to the DP001: CAR Barcoded Antibody spike-in Proxiome kit (v1.01) manual. The standard 155-plex PNA panel was supplemented with a specific anti-FMC63 probe to enable direct detection and spatial mapping of the CD19 CAR transgene. Samples were sequenced on an Illumina NextSeq 2000 system using 44 cycles for read 1 and 78 cycles for read 2.
Data processing
The generated sequencing data was processed by Pixelator 0.24.0 using nf-core/pixelator 3.0.1 with default parameters.
File Download
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In order to process the dataset FASTQ files, you need to have the required infrastructure by nf-core/pixelator.
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| Panel file | Size | Checksum (MD5) |
|---|---|---|
| Proxiome Immuno 155 +FMC63 Panel | 8.02 KiB | d4151dd768a6c7f1b31774933b20e8d2 |
| Samplesheet | Size | Checksum (MD5) |
|---|---|---|
| CART/Raji Coculture Samplesheet CSV | 2.04 KiB | 791f4920e28bbc8f2e6c995e5cbcbd9e |
| Input files | Size | Checksum (MD5) |
|---|---|---|
| PNA065 CARTcells S01 R1 FASTQ | 4.03 GiB | 2124ddec98e7bd082dbc9b1906a5d9fb |
| PNA065 CARTcells S01 R2 FASTQ | 6.88 GiB | 98c1b43d5306ae4dafe30e2eb95b8008 |
| PNA065 CARTcells Raji 4hrs 1to1 S02 R1 FASTQ | 3.55 GiB | 3fdb80a979bf31c5d5313a6552710709 |
| PNA065 CARTcells Raji 4hrs 1to1 S02 R2 FASTQ | 6.03 GiB | c6c5752f58fb50b2e31d4eb8d42fda83 |
| PNA065 CARTcells Raji 4hrs 5to1 S03 R1 FASTQ | 3.08 GiB | 594c9873eec22ab39a5002bffbc17525 |
| PNA065 CARTcells Raji 4hrs 5to1 S03 R2 FASTQ | 5.03 GiB | 5d39e2674d10c30d8e422d6e677afcd2 |
| PNA065 Raji S04 R1 FASTQ | 4.17 GiB | b84bbb9467f0b7cad376cdf0cdb9f1e7 |
| PNA065 Raji S04 R2 FASTQ | 7.18 GiB | d63f4d84b4d2b4cffe5a9f4ea47bf552 |
| PNA065 CARTcells Raji 24hrs 1to1 S05 R1 FASTQ | 3.99 GiB | 9209905cde280d4b8ac5e8f9ace7114f |
| PNA065 CARTcells Raji 24hrs 1to1 S05 R2 FASTQ | 6.59 GiB | 7169ff134b1f6b75e9d88a61424691c9 |
| PNA065 CARTcells Raji 24hrs 5to1 S06 R1 FASTQ | 4.78 GiB | 8482e9025e84d3f1abfff452ca88d9a7 |
| PNA065 CARTcells Raji 24hrs 5to1 S06 R2 FASTQ | 7.97 GiB | 81912d94a8063c9d6b0e3e34c4d80aad |
| PNA066 CARTcells S01 R1 FASTQ | 5.64 GiB | 48f8a1854c629e6da83e598149406df4 |
| PNA066 CARTcells S01 R2 FASTQ | 9.67 GiB | 16863c9351d0e04f24d19fd148965537 |
| PNA066 CARTcells Raji 4hrs 1to1 S02 R1 FASTQ | 5.5 GiB | adfc42bf0193df19d1a128b0f3ca7c5f |
| PNA066 CARTcells Raji 4hrs 1to1 S02 R2 FASTQ | 9.35 GiB | f7a16958815016c63c21d68364ea314d |
| PNA066 CARTcells Raji 4hrs 5to1 S03 R1 FASTQ | 4.55 GiB | cf47658617e415d31a5c4c78fa15633f |
| PNA066 CARTcells Raji 4hrs 5to1 S03 R2 FASTQ | 7.78 GiB | 73bc99f25cafc29f7f354d7b33ca0e1e |
| PNA066 Raji S04 R1 FASTQ | 4.33 GiB | abe04334f6ab3b6da8ca9deebbf008b2 |
| PNA066 Raji S04 R2 FASTQ | 7.47 GiB | fcfe823e880b41e443c86e03fafb7394 |
| PNA066 CARTcells Raji 24hrs 1to1 S05 R1 FASTQ | 5.83 GiB | 9786250b037ace922d630cb70b86d07d |
| PNA066 CARTcells Raji 24hrs 1to1 S05 R2 FASTQ | 9.95 GiB | 9481439d4ae7354dc052d88e9c927ae4 |
| PNA066 CARTcells Raji 24hrs 5to1 S06 R1 FASTQ | 5.78 GiB | 023c374321b5e8a718e823946bbd5f53 |
| PNA066 CARTcells Raji 24hrs 5to1 S06 R2 FASTQ | 9.93 GiB | 3897a1ce846497ff318d2411f80738be |
| Output files | Size | Checksum (MD5) |
|---|---|---|
| PNA065 CARTcells S01 PXL | 2.46 GiB | c180e86a2836eb20708e624a77f7b44a |
| PNA065 CARTcells Raji 4hrs 1to1 S02 PXL | 2.7 GiB | dadd0d1f98dce883ad563666bfb7dc59 |
| PNA065 CARTcells Raji 4hrs 5to1 S03 PXL | 1.18 GiB | 849325b9d9d655ebfb474c69efafce07 |
| PNA065 Raji S04 PXL | 3.2 GiB | fd86bd0fcde564ffcd00d2a158670f77 |
| PNA065 CARTcells Raji 24hrs 1to1 S05 PXL | 2.89 GiB | f0395c07da565ba60234a703a027ee55 |
| PNA065 CARTcells Raji 24hrs 5to1 S06 PXL | 2.84 GiB | a52aad5f3d10c5dd7694314628df6b09 |
| PNA066 CARTcells S01 PXL | 2.33 GiB | 2fcbcbcdafb98662ab128bfa867f935e |
| PNA066 CARTcells Raji 4hrs 1to1 S02 PXL | 2.75 GiB | 342472e7ff5a476b211e252b632586eb |
| PNA066 CARTcells Raji 4hrs 5to1 S03 PXL | 1.11 GiB | 96f1addbee75ae23b2a7efd8f8840bdc |
| PNA066 Raji S04 PXL | 2.51 GiB | 083d62d5576f429dc033e628610492d7 |
| PNA066 CARTcells Raji 24hrs 1to1 S05 PXL | 2.78 GiB | cb9dd33e47f38f317a14368737ec16ab |
| PNA066 CARTcells Raji 24hrs 5to1 S06 PXL | 2.61 GiB | c717af1afeac9ea144ca7870b11c6a02 |
| Proxiome Experiment summary | Size | Checksum (MD5) |
|---|---|---|
| PNA065 experiment summary | 112.41 MiB | 773a473d6a77aac610f96dc9a605db19 |
| PNA066 experiment summary | 108.54 MiB | 4dae7bf5d87e11b0ea36f515c491f40d |
# Panel file
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/proxiome-immuno-156-FMC63-v1.csv
# Samplesheet
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/samplesheet.CART-Raji-Coculture.csv
# 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_Raji_S04_S4_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA065_Raji_S04_S4_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_Raji_S04_S4_R1_001.fastq.gz
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/fastqs/PNA066_Raji_S04_S4_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
# Output files
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-33-74cd2a1a/pixelator/PNA065_CARTcells_S01.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-33-74cd2a1a/pixelator/PNA065_CARTcells_Raji_4hrs_1to1_S02.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-33-74cd2a1a/pixelator/PNA065_CARTcells_Raji_4hrs_5to1_S03.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-33-74cd2a1a/pixelator/PNA065_Raji_S04.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-33-74cd2a1a/pixelator/PNA065_CARTcells_Raji_24hrs_1to1_S05.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-33-74cd2a1a/pixelator/PNA065_CARTcells_Raji_24hrs_5to1_S06.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-13-24772739/pixelator/PNA066_CARTcells_S01.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-13-24772739/pixelator/PNA066_CARTcells_Raji_4hrs_1to1_S02.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-13-24772739/pixelator/PNA066_CARTcells_Raji_4hrs_5to1_S03.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-13-24772739/pixelator/PNA066_Raji_S04.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-13-24772739/pixelator/PNA066_CARTcells_Raji_24hrs_1to1_S05.layout.pxl
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-13-24772739/pixelator/PNA066_CARTcells_Raji_24hrs_5to1_S06.layout.pxl
# Proxiome Experiment summary
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-33-74cd2a1a/pixelator/experiment-summary.html
curl -O https://pixelgen-technologies-datasets.s3.eu-north-1.amazonaws.com/pna-datasets/v2/sha-4acd966-2026-04-13_15-55-13-24772739/pixelator/experiment-summary.html
License
Creative Commons Attribution-ShareAlike 4.0 International (CC BY-SA 4.0)
How to Cite
Single-Cell Protein Interactomes by the Proximity Network Assay. Filip Karlsson, Michele Simonetti, Christina Galonska, Max Karlsson, Hanna van Ooijen, Tomasz Kallas, Divya Thiagarajan, Maud Schweitzer, Ludvig Larsson, Vincent van Hoef, Pouria Tajvar, Johan Dahlberg, Florian De Temmerman, Louise Leijonancker, Sylvain Geny, Rikard Forlin, Erika Negrini, 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