γδ T cells were isolated from activated PBMCs using EasySep Human Gamma/Delta T Cell Isolation Kit (StemCell Technologies), according to the manufacturer’s instructions. The isolated supernatant was then resuspended in supplemented RPMI1640 media before use. To confirm isolation purity, cells were analysed by flow cytometry and γδ T cells identified as CD3+ αβTCR- (Supplementary Table 1 Supplementary Figure 1 Supplementary Figure 1
Easysep human gamma delta t cell isolation kit
Manufactured by STEMCELL
The EasySep Human Gamma/Delta T Cell Isolation Kit is a magnetic cell separation system designed to isolate human gamma/delta T cells from peripheral blood, leukapheresis samples, or other single-cell suspensions. The kit uses magnetic particles to label and isolate the target cells, allowing for a fast and convenient cell separation process.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque, by GE Healthcare (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Zoledronate, by Merck Group (1 mentions)
Recombinant human interleukin 2, by Thermo Fisher Scientific (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Recombinant human interleukin il 2, by Thermo Fisher Scientific (1 mentions)
Easysep human nk cell enrichment kit, by STEMCELL (1 mentions)
X vivo 15 media, by Lonza (1 mentions)
Human male ab serum, by Merck Group (1 mentions)
5 protocols using easysep human gamma delta t cell isolation kit
1
Isolation and Purification of Gamma-Delta T Cells
2
Vγ9Vδ2 T Cell Isolation and Expansion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were isolated from the healthy volunteers’ whole blood (The First Affiliated Hospital of Jinan University), following standard Ficoll−Paque-based (GE Healthcare) density gradient centrifugation protocol. Vγ9Vδ2 T cells were generated with Zoledronate (Sigma-Aldrich) as described (40 (link)). Briefly, isolated PBMCs were suspended in RPMI 1640 medium supplemented with 10% FBS, 100 U ml−1 recombinant human interleukin-2 (Peprotech), and Zoledronate (50 μM). The PBMCs were seeded into 24 well plates, followed by routine culture procedures for 10 days before further tests. Vγ9Vδ2 T cells were maintained at a cell density of 1×106 ml−1. When ratio of Vγ9Vδ2 T cells out of total CD3+ cells reached 90%, they could be used in experiments. Vγ9Vδ2 T cells were characterized with PerCP-conjugated antihuman TCR Vδ2 (BioLegend) and V500-conjugated antihuman CD3 (BD Biosciences) via flow cytometry. In some experiments, the Vγ9Vδ2 T cells were further purified by negative selection with EasySep™ Human Gamma/Delta T Cell Isolation Kit (STEM CELL), and the purity of enriched Vγ9Vδ2 T cells was validated by flow cytometry and was generally higher than 98%.
3
Expansion of Vδ2+ T Cells Using Zoledronic Acid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Zoledronic acid monohydrate ZOL (MCE, HY-13777A)-expanded Vγ9 Vδ2+ (Vδ2+) T cells were generated as described below. Human PBMCs (2.5 × 106/mL) were cultured in RPMI 1640 (Gibco, 11875093) medium supplemented with 10% fetal bovine serum (FBS; Gibco, 16140071), 2 mM L-glutamine, antibiotics, and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. ZOL (50 μM ZOL and 400 U/mL recombinant human interleukin-2 (rhIL-2; Peprotech, 200-02-50) were added on day 0. rhIL-2 was added at a final concentration of 100 U/mL once every two days from day 3, followed by routine culture procedures for 10 days before further experiments. When ratio of Vδ2+ T cells out to total CD3+ cells (Vδ2+/CD3+ T) reached 95%, they could be used in further experiments. ZOL-expanded Vδ2+ T cells were further purified by negative selection using the EasySep™ Human Gamma/Delta T Cell Isolation Kit (STEM CELL, 19255).
4
Expansion of Human Vγ9Vδ2 T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood from healthy male or female donors was obtained from the Guangzhou Blood Center. Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque-based (GE Healthcare, 17-1140-02) density gradient centrifugation. Zoledronate (ZOL) (Sigma, SML0223) expanded Vγ9Vδ2 T (Vδ2 T) cells were generated as described following.28 (link) Briefly, human PBMCs (2–3×106/mL) were cultured in RPMI 1640 (Gibco, 11875093) medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco, 16140071). Recombinant human interleukin (IL)-2 (Peprotech, 200-02-50) 400 U/mL and ZOL (50 µM) was added at day 0. Recombinant human IL-2 was added to a final concentration of 100 U/mL once every 2 days from day 3. T cells were maintained at a cell density of 1–2×106/mL until the percentage of Vγ9Vδ2/CD3 T is >90%. The purity of Vγ9Vδ2 T cells was determined by flow cytometry (BD VERSE). After 10–12 days of culture, cells were used in experiments. In some experiments, the Vγ9Vδ2 T cells were further purified by negative selection with EasySep Human Gamma/Delta T Cell Isolation Kit (STEM CELL, 19255). Unless mentioned otherwise, Vγ9Vδ2 T cells used in all the experiments were pretreated with 1α,25(OH)2D3 (50 nM) or vehicle two times at 1-day intervals.
5
Isolation and Activation of NK and γδ T Cells
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!