Freshly excised 3 mm punch biopsies from 3 BCC lesions were collected in DMEM media. Tissue were transported to cell culture lab on ice and stored in DMEM media for 24–48 h at 4 °C. Cell suspensions were generated according to the following protocol87 (link). Cells were processed for surface labeling with anti-CD3, anti-CD45, anti-CD4, and anti-CD8 antibodies. Live cells are distinguished from dead cells by using the fixable dye eFluor506 (eBioscience, Thermo Fisher Scientific, MA, USA). They were further permeabilized using a FOXP3 fixation and permeabilization kit (eBioscience, Thermo Fisher Scientific, MA, USA) and stained for Ki-67, FOXP3, and granzymeB. Data were acquired using the Aurora Five Laser flow cytometer (Cytek Biosciences, CA, US). Data were analyzed with FlowJo software version 10.5.3. (Tree Star Inc. OR, USA)88 .
Sahu A., Kose K., Kraehenbuehl L., Byers C., Holland A., Tembo T., Santella A., Alfonso A., Li M., Cordova M., Gill M., Fox C., Gonzalez S., Kumar P., Wang A.W., Kurtansky N., Chandrani P., Yin S., Mehta P., Navarrete-Dechent C., Peterson G., King K., Dusza S., Yang N., Liu S., Phillips W., Guitera P., Rossi A., Halpern A., Deng L., Pulitzer M., Marghoob A., Chen C.S., Merghoub T, & Rajadhyaksha M. (2022). In vivo tumor immune microenvironment phenotypes correlate with inflammation and vasculature to predict immunotherapy response. Nature Communications, 13, 5312.
Anti antibodies Anti cd3 Biopsies Cell cultureCells Tissue Tree
Corresponding Organization : Memorial Sloan Kettering Cancer Center
Other organizations :
Swim Across America, Parker Institute for Cancer Immunotherapy, Northeastern University, SUNY Downstate Health Sciences University, Caliber Imaging and Diagnostics (United States), Universidad de Alcalá, Icahn School of Medicine at Mount Sinai, Tata Memorial Hospital, Melanoma Institute Australia, Cornell University
Surface labeling with anti-CD3, anti-CD45, anti-CD4, and anti-CD8 antibodies
Staining for Ki-67, FOXP3, and granzymeB
control variables
Tissue transport to cell culture lab on ice
Storage in DMEM media for 24–48 h at 4 °C
Live cells distinguished from dead cells using the fixable dye eFluor506
Permeabilization using a FOXP3 fixation and permeabilization kit
controls
No positive or negative controls explicitly mentioned
Annotations
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