CD8+ T Cells were extracted from PBMCs isolated from whole blood using the EasySepTM Human CD8+ T cell Enrichment according to manufacturer’s protocol. Briefly, PBMCs were suspended in EasySepTM buffer (Cell Separation Buffer) (Stemcell) in a 14mL round bottom centrifuge tube (Corning). 50 μL/mL of Enrichment Cocktail was added to PBMCs and allowed to incubate at room temperature for 10 minutes. Magnetic particles were mixed by vortexing for 30 seconds. 150 μL/mL of magnetic particles were added to the PBMCs and allowed to incubate for 5 minutes at room temperature. The PBMC cocktail was topped up to 5 mL using EasySepTM Buffer. The PBMC cocktail was added to “The Big Easy” magnet (Stemcell) and allowed to incubate at room temperature for 5 minutes. The CD8+ T cells are the cells that have no bound magnets. These were poured into a fresh 15 mL conical and centrifuged at 1200 rpm for 5 minutes and plated in T cell media containing RPMI 1640 (Invitrogen), 10% fetal calf serum, β-mercaptoethanol (50 μM), 1% Pennecillin/Streptomycin, 1% Insulin-tellurium-selenium (Thermofisher), IL-2 (30 U/mL) (Thermofisher) and IL-7 (0.5 ng/mL) (Thermofisher) [9 (link)].
Big easy magnet
Manufactured by STEMCELL
The Big Easy magnet is a laboratory equipment designed for cell separation applications. It provides a strong magnetic field to facilitate the separation of magnetically labeled cells from a cell suspension.
Automatically generated - may contain errors
Lab products found in correlation
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
Easysep direct human neutrophil isolation kit, by STEMCELL (2 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Easysep buffer, by STEMCELL (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Easysep human cd8 t cell enrichment kit, by STEMCELL (1 mentions)
Phosphate buffered saline pbs, by Eurobio Scientific (1 mentions)
Countess 3 fl automated cell counter, by Thermo Fisher Scientific (1 mentions)
Easysep human cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Ficoll paque, by Merck Group (1 mentions)
Cd3 cd28 coated dynabeads, by Thermo Fisher Scientific (1 mentions)
Easysep human t cell isolation kit, by STEMCELL (1 mentions)
Edta vacutainer tube, by BD (1 mentions)
Polystyrene t25 flasks, by Corning (1 mentions)
Calcein red orange am, by Thermo Fisher Scientific (1 mentions)
8 protocols using big easy magnet
1
Isolation of CD8+ T Cells from PBMCs
2
Enrichment and Labeling of CD8+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human peripheral blood mononuclear cells (PBMC) were purchased from Cellular Technology Limited (CTL, Cleveland, OH, USA). For experiments using enriched CD8+ T cells, total PBMC were thawed and CD8+ T cells enriched using EasySep™ Human CD8+ T Cell Enrichment Kit (Stemcell Technologies Inc, Catalog # 17953). Magnetic separation was done with The Big Easy magnet (StemCell Technologies; Catalog #18001) as per manufacturer’s instructions. Purified CD8α+ cells were stained CFSE (carboxyfluorescein diacetate succinimidyl ester from Tonbo Biosciences; SKU: 13-0850) at a concentration of 5 μM per manufacturer’s protocol or with CellTrace Violet (Thermofisher) following manufacturer’s protocol.
3
Magnetic isolation of granulocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IG and Mature granulocytes (MG) were obtained by negative magnetic sorting from PB using the EasySep Direct Human Neutrophil Isolation kit (STEMCELL). 5 mL of PB was incubated for 5 min at room temperature with an isolation cocktail and RapidSphere from the kit according to the manufacturer’s instruction. Phosphate buffered saline (PBS) (Eurobio Scientific) was added to a final volume of 12 mL, and the tube was placed in the “The Big Easy” magnet (STEMCELL) for 10 min, before pouring the granulocyte containing supernatant into a new tube. The process was repeated twice to achieve high purity of the granulocytic population. A lower cutoff of granulocytes (IG or MG) concentration was set at 90% among total cells collected as quantified by flow cytometry. Isolated granulocytes were counted using a Countess 3FL Automated cell Counter (Invitrogen).
4
Activation and Differentiation of CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human peripheral blood mononuclear cells (PBMC) were purchased from Cellular Technology Limited (CTL, Cleveland, OH, USA). CD4+ T cells were enriched using EasySep™ Human CD4+ T Cell Enrichment Kit (Stemcell Technologies Inc, Catalog # 17952). Magnetic separation was done with The Big Easy magnet (StemCell Technologies; Catalog #18001) as per manufacturer’s instructions. Thermo Scientific IMMULON II Flat Plate 96-well (Fisher 1424561LC) were coated with anti-CD3 antibody (clone: OKT3, Tonbo; 10 μg/mL in PBS) overnight at 4°C. Plates were washed once with RPMI/10% FCS media prior to plating T cells. Stimulation cultures of T cells included addition of anti-CD28 (clone: CD28.2, Tonbo; 1 μg/mL) and IL-2 (Tonbo; 100 U/mL) in complete RPMI media. Enriched CD4+ cells were spun down, plated at 8 × 104 cells/well into anti-CD3-coated plates (with anti-CD28 and IL-2), and allowed to differentiate for 4–5 days in the presence of vehicle, indoximod (1.5–100 μM), Kyn (50–100 μM) and/or AhR inhibitor GNF351 (500 nM) or CH223191 (10 μM).
5
Isolation and Transduction of Primary T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood from healthy donors was obtained from apheresis leukoreduction collars (Brigham and Women's Hospital, Crimson Core). Peripheral blood mononuclear cells were extracted using centrifugation (35 minutes, 400 × g) through Ficoll Paque (Sigma-Aldrich, GE17–1440–02). T cells were isolated using the EasySep Human T-cell Isolation Kit (StemCell Technologies, 17951) and the Big Easy magnet (StemCell, 18001), according to the manufacturer's protocol. T cells were stimulated with CD3/CD28-coated Dynabeads (Thermo Fisher Scientific, 11131D) and maintained in R10 supplemented with 20 U/mL IL2. 24–30 hours after isolation, T cells were infected with MSLN CAR by adding lentivirus dropwise to each well at an MOI of 5–6; infection efficiencies ranged from approximately 50% to 75%. 7 days after isolation, Dynabeads were removed; 14 days after isolation, T cells were frozen in 90% FBS supplemented with 10% DMSO. Fifteen to 20 hours before plating a coculture, T cells were thawed and cultured in R10 sans IL2.
6
Isolation and Staining of Human Neutrophils
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All blood specimens from patients were obtained with informed consent according to the institutional review board-approved study protocol at the University of California - San Francisco (Study #21–35147), see Table S2 for demographic information. Fresh samples of peripheral blood from healthy adult volunteers were collected via a 23-gauge butterfly needle collection set (BD #23–021-022) into 10 ml Vacutainer EDTA tubes (BD #366643). Blood was kept on a shaker at minimum setting and utilized within 2 hours of the draw. Neutrophils were isolated using the EasySep Direct Human Neutrophil Isolation Kit (STEMCELL Tech #19666) with the BigEasy magnet (STEMCELL Tech #18001) according to the manufacturer’s protocol.
Isolated neutrophils were spun down at 200g for 5 min and resuspended in a dye media consisting of imaging media containing 5ug/ml Hoechst 3334 (Invitrogen #H3570) and 0.25 uM Calcein Red-Orange AM (Invitrogen #C34851). This cell suspension was incubated at room temperature in the dark for 15 min, and then the cells were spun down at 200g for 5 min. The dye medium was aspirated and replaced with R10 to achieve a final cell density at or below 1×106 cells/mL. Purified neutrophils were then kept in polystyrene T25 flasks (Corning) at 37°C in a 5% CO2 environment until imaging. Cells were used ~5–8 hours post-isolation.
Isolated neutrophils were spun down at 200g for 5 min and resuspended in a dye media consisting of imaging media containing 5ug/ml Hoechst 3334 (Invitrogen #H3570) and 0.25 uM Calcein Red-Orange AM (Invitrogen #C34851). This cell suspension was incubated at room temperature in the dark for 15 min, and then the cells were spun down at 200g for 5 min. The dye medium was aspirated and replaced with R10 to achieve a final cell density at or below 1×106 cells/mL. Purified neutrophils were then kept in polystyrene T25 flasks (Corning) at 37°C in a 5% CO2 environment until imaging. Cells were used ~5–8 hours post-isolation.
7
CFSE-Based Mixed Leukocyte Reaction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
First, splenocytes isolated from WT mice were labeled with 2 µM CFSE, at 37°C for 15 min. For mixed leukocyte reaction, WT splenocytes labeled with CFSE (5 × 105/well) were cocultured with DCs isolated from F1 mice by the CD11c immunomagnetic separation kit (105/well, Big Easy Magnet; STEMCELL Technologies) in triplicate in 96-well plates containing complete DMEM for 72 h. After 72 h, the cells were harvested, and the cell proliferation was measured by flow cytometric analysis of CFSE dilution in the following groups: F1 splenocytes + F1 DCs (negative control group), WT splenocytes + F1 DCs + vehicle, and WT splenocytes + F1 DCs + zileuton (40 µM).
8
Isolation of T Cell Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All T cells were isolated “un-touched” using negative selection kits by STEMCELL. Human Naïve CD8 (19258), Naïve CD4 (19555), Memory CD4 (19157), Memory CD8 (19159), Total CD4 (17952), Total CD8 (17953), and Total CD3+ T (17951) cell kits were routinely used. When using other brands, caution must be taken before use to ensure the absence of FcR-blocking reagents. Negative-selection kits enrich for all αβ T cell subsets by depleting markers associated with “unwanted” cells (e.g., CD14, CD16, CD19, CD20, CD34, CD36, CD56, CD61, CD66b, CD123, TCRγ/δ, glycophorin A). Naïve T cell negative-selection and Memory T cell negative-selection kits deplete markers to enrich for αβ T cells in addition to depleting other markers associated with either memory/activated (CD45RO, CD57, CD94, CD244, CD25) or naïve T cells (CD45RA), respectively. Mouse naïve T cells (19848), Total B cells (19854), and Total T cells (19851) kits were also used. CD14+ monocytes and CD56+ NK Cells were isolated from peripheral blood using positive selection kits (17858 and 17855, respectively). All labeled cells were separated using the EasySep™ or Big Easy™ magnet (STEMCELL).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!