To determine whether the PD-L1 CAR-engineered Jurkat T cells bound to PD-L1 presented on the tumor cell surface to promote cell aggregation, PD-L1-positive K562 tumor cells labeled with Cell Proliferation Dye eFluor 670 (Invitrogen #65–0840) were mixed with PD-L1 CAR-modified Jurkat T cells or untransduced Jurkat T cells labeled with CellTrace CFSE (Invitrogen #C34554) at an effector: target (E: T) ratio of 1:1 in a 1.5 mL eppendorf tube at room temperature (RT) for 1 h. The proportion of cells forming heterologous cell aggregates (eFluor 670+CFSE+) was assessed by flow cytometry (BD LSRFortessa).
Efluor 670
Manufactured by BD
Sourced in United States
EFluor 670 is a fluorescent dye used for cell proliferation and viability assays. It is a stable, cell-permeant dye that binds to cellular proteins, allowing for the tracking of cell division and the identification of proliferating cells. The dye has an excitation wavelength of 647 nm and an emission wavelength of 670 nm, making it compatible with common fluorescence detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Texas red phalloidin, by Thermo Fisher Scientific (2 mentions)
Phalloidin af488, by Thermo Fisher Scientific (2 mentions)
Anti cd45 apc, by BD (2 mentions)
Anti fak, by Abcam (2 mentions)
Anti ve cadherin, by Abcam (2 mentions)
Anti vinculin, by Santa Cruz Biotechnology (2 mentions)
Anti cd45ro, by BD (2 mentions)
Anti ccr7, by BD (2 mentions)
Anti tim3, by BD (2 mentions)
Anti alcam pe, by Abcam (2 mentions)
Anti icam1 pe, by Abcam (2 mentions)
Anti vcam1 pe, by Abcam (2 mentions)
Anti chs1, by Abcam (2 mentions)
Anti hif 1, by BD (2 mentions)
Anti pzap 70 fitc, by BD (2 mentions)
Anti pzap 70 pe, by BD (2 mentions)
Anti chs percp, by BD (2 mentions)
Anti talin 1, by Santa Cruz Biotechnology (2 mentions)
Goat anti human fc pe, by Merck Group (2 mentions)
Anti fab dylight 488, by Jackson ImmunoResearch (2 mentions)
6 protocols using efluor 670
1
PD-L1 CAR-Jurkat T Cell Binding
2
Multimodal Analysis of Cellular Adhesion Proteins
3
Activated T Cell Phenotyping by Flow Cytometry
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Flow cytometry was performed on a FACS LSR II or Fortessa (BD Biosciences). For lymphocyte activation studies, soluble anti-CD3 (500 ng/ml; clone OKT3, Bio X Cell) ± anti-CD28 (1 μg/ml; clone CD28.2, BD Biosciences) or IL-2 (PeproTech) was added to media for 2 days. Proliferation was measured by CFSE (eFluor 670, BD Biosciences) after 3 days. EVs were added every 24 hours (5 μg/ml). PD1 blockade was achieved by adding anti-PD1 (10 μg/ml; EH12) or immunoglobulin G control (clone MOPC-21, Bio X Cell). Cells were gated by FSC (forward scatter)/SCC (side scatter) while excluding duplets by FSC-A/FSC-H. Subsequently, CD3+CD56− T cells were gated with further classification of CD4+ and CD8+ T cells. Finally, CD4+ and CD8+ T cells were measured for their CD69, CD25, PD1, and TIM3 expression (seen as a downloadable figure). Cells were stained with the following antibodies: CD3/PE-Cy5.5 (1:100; clone SK7, eBioscience), CD4/FITC (1:100; clone RPA-T4, BD Biosciences), CD8/AmCyan (1:10; clone SK1, BD Biosciences), CD16/APC-Cy7 (1:100, BD Biosciences), CD25/PE-Cy7 (1:10; clone BC96, eBioscience), CD56/V450 (1:30; clone B159, BD Biosciences), CD69/APC (1:10; clone FN50, BD Biosciences), TIM3/APC-Cy7 (1:100, clone F38-2E2, BD Biosciences), and PD1/PE (1:100; clone J105, eBioscience).
4
Analyzing Treg-Mediated CD4+ T Cell Inhibition
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For T cell inhibition assays, CD4+ FOXP3+ (eGFP+) Tregs were isolated from CRC lesions from Foxp3/eGFP mice and divided into ST2+ or ST2− cell populations using a FACSAria II cell sorter (BD Biosciences). As responder cells, CD4+ T cells were MACS-purified from spleens of naive St2−/− mice (BALB/c background) using a CD4+ T Cell Isolation Kit II (Miltenyi Biotec) and labeled with eFluor670 (Thermo Fisher) following the manufacturer’s instructions. eFluor670-labeled responder CD4+ T cells (5 × 104) were either cultured alone or co-cultured with CD4+ FOXP3+ (eGFP+) ST2+ or ST2− Tregs (2.5 × 104) for 3 days in the presence of 1 μg/ml anti-CD3 (clone 145-2C11; BD Biosciences). Irradiated splenocytes from naïve St2−/− mice served as antigen-presenting cells (1.5 × 105). Where indicated, 30 ng/ml rmIL-33 (BioLegend) was added to the culture.
5
Treg Suppression of CD4+ T Cell Proliferation
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CD4+CD25+ nTreg and CD4+CD25− non-Treg T cells were sorted from indicated strains. Treg were labelled with CFSE (Life Technologies) and responder non-Treg with eFluor 670 (Molecular Probes, Eugene, OR). Responder CD4+CD25− T cells (5 × 104 cells per well) were cultured with or without Treg at responder:Treg ratios of 1:0, 1:1, 2:1, 4:1 and 8:1 with irradiated (800 rad) syngeneic T cell-depleted splenocytes (5 × 104 cells per well) in 96-well U-bottom plates (Corning) and anti-CD3ɛ mAb (1 μg ml−1, clone 145-2C11, eBioscience, 16-0031) for 3 days. Cells were collected, and cell division was measured by assessing relative eFluor 670 dilution on an LSRFortessa cytometer (BD).
6
Multimodal Analysis of Cellular Adhesion Proteins
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Anti-FAK, anti-ALCAM-PE, anti-ICAM1-PE, anti-VCAM1-PE, anti-VE cadherin, anti-von Willebrand (vWF) factor, anti-CHS1 were purchased from Abcam (Cambridge, MA, USA). OX124, mouse anti-human monoclonal antibody against HS, was a kind gift from Dr. Marion Brown, Oxford, UK. Biotin-labeled Anti-CD18 KIM127 was purchased from Exploratory Research Cell Tech Therapeutics Ltd. (Slough, UK). Anti-phospho-SLP-76; pTyr128, anti-VE-cadherin were purchased from Cell signaling Technologies (Danvers, MA, USA). Anti-HIF-1, anti-pZAP-70-FITC, anti-pZAP-70-PE, anti- LAG3, anti-TIM3, anti-PD-1, eFluor 670, anti-CD45-APC, anti-CHS-PerCP, anti-CCR7, anti-CD45RO were purchased from BD biosciences (Franklin Lakes, NJ, USA). Anti-Talin-1 and anti-Vinculin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Goat anti-human Fc-PE was purchased from Millipore (Billerica, MA, USA). Alexa Fluor (AF) labelled secondary anti-mouse, anti-goat anti-rabbit antibodies, streptavidin AF 488 antibody; Texas Red Phalloidin and AF488 Phalloidin were purchased from Life Technologies (Carlsbad, CA, USA). Anti-Fab DyLight 488 was purchased from Jackson ImmunoResearch (Suffolk, UK)
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