Tumor single cell suspensions from were thawed and CD45+ cells were enriched using EasySep™ Release Human CD45 Positive Selection Kit (StemCell Technologies, cat#100–0105) based on manufacturer’s protocol. We then added Fc block to the isolated cells and incubated RT for 5mins, then stained them with fluorescently labelled antibodies antibodies CD3, CD4, PD-1, CD25, CD127, CD200, CXCR6 at 4 °C for 30mins. Next, we sorted the different CD4 T cell subsets (Effectors, TFH, Bystanders) using BD FACSymphony S6 System and cultured the sorted cells overnight in CTL media supplemented with IL-2 50U/ml and IL-7 5ng/ml. After resting the cells overnight, T cells were labelled using CellTrace Violet Cell Proliferation Kit (Thermofisher Scientific, cat# C34571) per manufacturer’s recommendations. Next, we added CD3/CD28 Dynabeads to the labelled cells at 3:1 bead to cell ratio in CTL, and cultured cells at 37 °C. After 24hours, IL-2 10U/ml was added to the cells and cultured for 4 days. On day 4, we removed the Dynabeads and stained cells with antibodies CD3 BV711, CD4 BUV395, CD19 PE and Proliferation was analyzed using flow cytometry. Relative proliferation of cell fractions was compared between patients using a ratio paired t-test.
Easysep release human cd45 positive selection kit
Manufactured by STEMCELL
Sourced in Canada
The EasySep™ Release Human CD45 Positive Selection Kit is a laboratory tool designed to isolate CD45-positive cells from human samples. It utilizes magnetic particles and a specialized buffer system to selectively bind and separate the desired cell population.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet cell proliferation kit, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 fgf2, by Thermo Fisher Scientific (1 mentions)
Brainphys neuronal medium, by STEMCELL (1 mentions)
Transforming growth factor β1 tgf β1, by Thermo Fisher Scientific (1 mentions)
Stemdiff cerebral organoid kit, by STEMCELL (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Heparin, by Merck Group (1 mentions)
Poly d lysine hydrobromide, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Legendplex human cd8 nk panel 13 plex, by BioLegend (1 mentions)
3 protocols using easysep release human cd45 positive selection kit
1
Purification and proliferation of CD4+ T cells
2
Cerebral Organoid Culture Protocol
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L-ascorbic acid (LAA), ethylenediaminetetraacetic acid (EDTA), heparin, laminin from Engelbreth-Holm-Swarm murine sarcoma, Lowry assay kit (Peterson’s modification), poly-D-lysine hydrobromide, protease inhibitor cocktail (Cat#P8340), sodium selenite, and Terg-A-Zyme detergent were purchased from Millipore Sigma (Oakville, ON, Canada). Transforming growth factor-β1 (TGF-β1; cat #100–21) and fibroblast growth factor 2 (FGF2; cat#100-18B) were purchased from Peprotech (Cranbury, NJ, United States) was purchased from Peprotech (Cranbury, NJ, United States). Recombinant human transferrin (cat#777TRF029) was purchased from InVitria (Fort Collins, CO, United States). Radioimmunoprecipitation assay (RIPA) 10x buffer and the ROCK inhibitor Y-27632 (cat# 13624S) were purchased from Cell Signaling Technologies (Whitby, ON, Canada). BrainPhys™ Neuronal Medium, EasySep™ Release Human CD45 Positive Selection Kit, and the STEMdiff™ Cerebral Organoid Kit (cat#08570) were obtained from STEMCELL Technologies (Vancouver, BC, Canada). A full list of antibodies and their suppliers is provided in Table 2 . All other reagents were sourced from Fisher Scientific (Ottawa, ON, Canada).
3
Cytotoxic T Cell Stimulation Assay
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Tumour cells were seeded in complete RPMI medium one day prior to co-culture. Complete RPMI medium includes RPMI (Gibco 21870076), 10% fetal bovine serum, 1% l -glutamine, 1% penicillin-streptomycin. The next day, RPMI medium was replaced with T cell medium and antigen-specific T cells were seeded on top of the tumour cells at a 1:1 E:T ratio with IL-2 at 50 IU ml−1. Subsequent repeated co-cultures were set up every 48 h. For each co-culture, T cells were collected and counted using the Vi-CELL XR cell counter and viability analyser and replated onto fresh target tumour cells at a 1:1 E:T ratio. Before using the T cells for any assays, T cells were collected, counted and purified using EasySep Release Human CD45 positive selection kit (Stem Cell 100-0105) or purified by flow sorting. For ELISA experiments, after 5 stimulations of TCR T cells with target cells (A375), supernatant of co-cultures was collected and analyzed using the LEGENDplex Human CD8/NK Panel 13-plex (Biolegend 740267) according to the manufacturer’s instructions.
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