Spleens were harvested from naïve FVB mice and passed through 100 µm screens. CD8+T cells were isolated from splenocytes via immunomagnetic negative selection (StemCell #19853), and then labeled with carboxyfluorescein succinimidyl ester (Biolegend #423801) according to the manufacturer’s instructions. Tumors were collected from treated tumor-bearing mice on day 36, processed into single-cell suspensions as above, and CD11b+Gr-1+Ly-6G+MDSCs were isolated (Miltenyi Biotec #130-094-538). 1×105 labeled CD8+T cells were cultured together with MDSCs at a 1:1 ratio. CD8+T cells were stimulated with anti-CD3/anti-CD28 coated beads (Thermo Fisher 11 456D) at a ratio of 2 beads per CD8+T cell. Cells were cultured with 30 units/mL of human IL-2 for 72 hours in 96-well plates before analysis via flow cytometry.
Anti cd3 anti cd28 coated beads
Manufactured by Thermo Fisher Scientific
Sourced in Norway
Anti-CD3/anti-CD28-coated beads are a type of immunomagnetic bead used for T-cell activation and expansion. The beads are coated with antibodies that target the CD3 and CD28 receptors on the surface of T cells, which stimulates their proliferation and activation.
Automatically generated - may contain errors
Lab products found in correlation
Lymphocyte separation medium, by Lonza (3 mentions)
Easysep human na ve cd4 t cell enrichment kit, by STEMCELL (2 mentions)
Rpmi 1640 glutamax media, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Lonza (2 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Cfse labeled, by Thermo Fisher Scientific (1 mentions)
Akt1 2 inhibitor, by Merck Group (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Easysep human naive cd4 t cell enrichment kit, by STEMCELL (1 mentions)
Cloxacillin, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Gentamicin, by Merck Group (1 mentions)
Simulect, by Novartis (1 mentions)
Ro 25 1553, by Bachem (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Gallios cytometer, by Beckman Coulter (1 mentions)
Anti cd25 pecy7, by Beckman Coulter (1 mentions)
Anti hla dr pe cy5, by Beckman Coulter (1 mentions)
Anti human foxp3 staining set, by Thermo Fisher Scientific (1 mentions)
22 protocols using anti cd3 anti cd28 coated beads
1
Isolation and Functional Assay of Murine CD8+ T Cells and MDSCs
2
Intestinal Organoid Differentiation and Immune Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids were incubated in TrypLE Express (Thermo Fisher Scientific) and mechanically disrupted using a P1000 pipette. Organoid fragments were seeded on 0.4 μm pore‐size Transwell polyester (PET) membranes (Corning) in 24‐well plates, with a 6.5‐mm insert. Organoids were cultured for 8–10 days in normal organoid media (nondifferentiation media, NDM) to achieve a confluent monolayer, with media changed every 48 h. An EVOM TEER reader (World Precision Instruments, USA) was used to monitor monolayer development. Once monolayers were confluent (Ω > 800), monolayers were cultured in differentiation media (DFM, organoid media containing no Wnt‐3a or R‐spondin) for 72 h then indicated numbers of F. prausnitzii, matched‐patient immune cells, or anti‐CD3/anti‐CD28‐coated beads (Thermo Fisher Scientific) were added to mucosal (upper) or serosal (lower) Transwell compartments for a further 72 h.
3
In vitro Suppression Assay for Regulatory T Cells
4
Isolation and Activation of Naïve CD4+ T Cells
5
T Cell Activation Inhibitor Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Compounds were reconstituted at 10 mM with DMSO and diluted to 1 μM, and 0.1 μM for cell culture. Cells are washed twice prior to addition of inhibitors. 30–60 minutes after addition of inhibitors, T cells were re-stimulated with anti-CD3/anti-CD28 coated beads (Life Technologies, Grand Island, NY). Cells are incubated with inhibitor compounds and beads for 18–24 hours. AKT 1/2 inhibitor (cat# 612847-09-03) was purchased from EMD Millipore (Billerica, MA).
6
CD4+ T Cell ELISPOT Assay
7
Isolation and Activation of Naïve CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated by gradient centrifugation with Lymphocyte Separation Medium (Lonza) and cultured in RPMI 1640 medium supplemented with 10% FCS (Hyclone) plus Pen/Strep/Glutamine. Naïve CD4+CD45RA+ T cells were purified from fresh PBMCs using an EasySep™ Human naïve CD4 T Cell Enrichment Kit (STEMCELL Technologies). Purity of cell populations was consistently >95%.
To induce effector CD4+ T cells, CD4+CD45RA+ T cells were stimulated with anti-CD3/anti-CD28-coated beads (Life Technologies AS) at a ratio of 2:1 for 72 h.
To induce effector CD4+ T cells, CD4+CD45RA+ T cells were stimulated with anti-CD3/anti-CD28-coated beads (Life Technologies AS) at a ratio of 2:1 for 72 h.
8
Isolation and Activation of Naive CD4+ T Cells
9
Expansion of CD4+ Memory T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD4+CD45RO+ T cells were cultured at 10 × 104 and at 5 × 104 cells/well (for HD and eRA patients, respectively) in RPMI-1640-GlutaMAX media (Life Technologies, Carlsbad, CA, USA) supplemented with 10 % fetal bovine serum (Lonza, Basel, Switzerland) and 1 % penicillin/streptomycin (Life Technologies). Cells were activated/expanded with anti-CD3/anti-CD28 coated beads (Life Technologies). CD4+CD45RO+ T cells were cultured in the absence or presence of 10nM of VIP (Polypeptide group, Strasbourg, France) for both HD and eRA patients.
10
Investigating BXM's Impact on PBMC Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs from buffy coats were isolated on a Ficoll-Hypaque (Rafer, Madrid, Spain) density gradient and treated with BXM (Simulect, Novartis Pharma, Basel, Switzerland) to analyze its effect on CD4, CD8 and Treg viability, proliferation, Foxp3 expression and cytokine secretion. Briefly, CFSE-labeled PBMCs (CFSE from Life Technologies, Carlsbad, CA) were cultured with RPMI 1640 medium (Biochrome) supplemented with 10% heat-inactivated FCS, 500 U/ml IL-2 and a mix of antibiotics (125 µg/ml cloxacillin, 125 µg/ml ampicillin and 40 µg/ml gentamicin; Sigma-Aldrich, St. Louis, MO). CFSE-labeled PBMCs (Life Technologies) were stimulated with anti-CD3/anti-CD28-coated beads (Life Technologies) at 0.5:1 or 1:1 ratio (bead:PBMC), treated with 10 µg/ml of BXM (Simulect, Novartis Pharma) and cultured for 72 h in the presence of IL-2. This concentration was chosen considering the serum concentration that is reached in pediatric patients treated with BXM20 (link), and looking for maximal suppressive effect46 (link). After culture, all samples were analysed by flow cytometry.
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!