HTAPP_Brief method description_Fresh pediatric glioma tissue processing, FACS and single cell Smart-Seq2 v1 (protocols.io.bu6gnzbw)

protocols.io ◽  
2021 ◽  
Author(s):  
Bernhard Englinger ◽  
McKenzie L ◽  
Mario Suva ◽  
Mariella L
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jeremy A. Lombardo ◽  
Marzieh Aliaghaei ◽  
Quy H. Nguyen ◽  
Kai Kessenbrock ◽  
Jered B. Haun

AbstractTissues are complex mixtures of different cell subtypes, and this diversity is increasingly characterized using high-throughput single cell analysis methods. However, these efforts are hindered, as tissues must first be dissociated into single cell suspensions using methods that are often inefficient, labor-intensive, highly variable, and potentially biased towards certain cell subtypes. Here, we present a microfluidic platform consisting of three tissue processing technologies that combine tissue digestion, disaggregation, and filtration. The platform is evaluated using a diverse array of tissues. For kidney and mammary tumor, microfluidic processing produces 2.5-fold more single cells. Single cell RNA sequencing further reveals that endothelial cells, fibroblasts, and basal epithelium are enriched without affecting stress response. For liver and heart, processing time is dramatically reduced. We also demonstrate that recovery of cells from the system at periodic intervals during processing increases hepatocyte and cardiomyocyte numbers, as well as increases reproducibility from batch-to-batch for all tissues.


2021 ◽  
Author(s):  
Daniel Rainbow ◽  
Sarah Howlett ◽  
Lorna Jarvis ◽  
Joanne Jones

This protocol has been developed for the simultaneous processing of multiple human tissues to extract immune cells for single cell RNA sequencing using the 10X platform, and ideal for atlasing projects. Included in this protocol are the steps needed to go from tissue to loading the 10X Chromium for single cell RNA sequencing and includes the hashtag and CiteSeq labelling of cells as well as the details needed to stimulate cells with PMA+I.


2016 ◽  
Vol 18 (suppl_6) ◽  
pp. vi152-vi153
Author(s):  
Amanda Saratsis ◽  
Tina Huang ◽  
Andrea Piunti ◽  
Rintaro Hashizume ◽  
Elizabeth Bartom ◽  
...  

2017 ◽  
Vol 19 (suppl_4) ◽  
pp. iv9-iv10
Author(s):  
Tina Huang ◽  
Andrea Piunti ◽  
Rintaro Hashizume ◽  
Elizabeth Bartom ◽  
Jin Qi ◽  
...  

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi119-vi119
Author(s):  
Julie Messiaen ◽  
Pouya Nasari ◽  
Yannick Van Herck ◽  
Ben Verhaaren ◽  
Ivey Sebastian ◽  
...  

Abstract High-grade glioma are the main cause of cancer-related death in children. Despite extensive research, their prognosis remains poor with very few treatment options. This can be attributed to the highly heterogeneous and plastic nature of glioma tumor cells and their interactions with the microenvironment, although quantitative data are still largely missing. Here, we used high-dimensional, multiplexed immunohistochemistry to map the spatial, single-cell tissue architecture of 31 pediatric glioma samples covering 9 histologic diagnoses. This novel approach allowed us to map the spatial distribution of the various tumoral subtypes, which typically occur in specific tumoral niches, and how these interact with their local immune-microenvironment. Finally, by aligning these findings to the clinical data of the patients and comparing these to adult glioblastoma, we are now able to more precisely describe the heterogeneous landscape of pediatric glioma at single-cell resolution.


Author(s):  
Debby A. Jennings ◽  
Michael J. Morykwas ◽  
Louis C. Argenta

Grafts of cultured allogenic or autogenic keratlnocytes have proven to be an effective treatment of chronic wounds and burns. This study utilized a collagen substrate for keratinocyte and fibroblast attachment. The substrate provided mechanical stability and augmented graft manipulation onto the wound bed. Graft integrity was confirmed by light and transmission electron microscopy.Bovine Type I dermal collagen sheets (100 μm thick) were crosslinked with 254 nm UV light (13.5 Joules/cm2) to improve mechanical properties and reduce degradation. A single cell suspension of third passage neonatal foreskin fibroblasts were plated onto the collagen. Five days later, a single cell suspension of first passage neonatal foreskin keratinocytes were plated on the opposite side of the collagen. The grafts were cultured for one month.The grafts were fixed in phosphate buffered 4% formaldehyde/1% glutaraldehyde for 24 hours. Graft pieces were then washed in 0.13 M phosphate buffer, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in Polybed 812.


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