B16 cells overexpressing PD-L1 were treated overnight with 20 ng ml–1 murine IFN-γ (Preprotech), washed, coated with 1.0 μM desired protein (that is, OVA, Neo-2/15, Ctrl-Neo2A and so on) for 10 min, washed and resuspended repeatedly and replated in a U-bottom plate. Mouse trp1-specific CD8 T cells (JAX Stock, no. 030958) were isolated via a negative isolation kit (Stemcell, no. 19853) and plated under the listed conditions either alone or in coculture with B16 cells at a 1:1 ratio for 1 day. To confirm trans-activation, 1.0 μM αPD-L1 or Ctrl VHH was added at the start of coculture. T cells were then harvested 24–36 h later and analyzed by flow cytometry for expression of activation markers CD25 (Biolegend, no. 102017) and CD69 (Biolegend, no. 104508). T cells were gated separately from B16 cells via FSC/SSC gating and CD8 staining (Biolegend, no. 100728). All antibodies were used at 1:100 dilution.
Negative isolation kit
Manufactured by STEMCELL
Sourced in Canada
The Negative Isolation Kit is a lab equipment product designed to isolate cells of interest from a mixed cell population. It uses an indirect magnetic labeling system to negatively select the target cells, leaving the cells of interest unlabeled and free of modifications. The kit provides a simple and efficient method for cell isolation, preserving the natural state of the separated cells.
Automatically generated - may contain errors
Lab products found in correlation
Lymphocyte separation media, by Corning (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
Murine ifn γ, by Thermo Fisher Scientific (1 mentions)
Cell tracker violet, by Thermo Fisher Scientific (1 mentions)
Golgiplug, by BD (1 mentions)
Lsriib, by BD (1 mentions)
Anti pe beads, by Miltenyi Biotec (1 mentions)
Aquavivid, by Thermo Fisher Scientific (1 mentions)
Immunocult xf t cell expansion medium, by STEMCELL (1 mentions)
Immunocult human cd3 cd28 t cell activator, by STEMCELL (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Magnetic bead isolation, by STEMCELL (1 mentions)
Dynabeads human t activator cd3 28, by Thermo Fisher Scientific (1 mentions)
Celltrace violet, by Thermo Fisher Scientific (1 mentions)
Lsr 2 fortessa cytometer, by BD (1 mentions)
12 protocols using negative isolation kit
1
Analyzing CD8 T cell Activation by PD-L1
2
Lentiviral TCR Transduction of T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Buffy coat was obtained from local Shepard blood center. PBMCs were harvested by centrifugation on a Lymphoprep Ficoll gradient, diluted to 1 × 107 cells/ml, aliquoted, and frozen. Lentiviral vectors expressing TCRs were prepared by 4 a plasmid co-transfection as previously described (20 (link)–22 (link)). Total T cells were isolated from PBMC by negative isolation kit (STEMCELL Technologies, Vancouver, BC, Canada) and then transduced by lentiviral vectors at MOI of 20–40 as described (15 (link)). Between 12–15 days after transduction, tetramer (NIH Tetramer Core Facility) staining was conducted to measure the percentage of TCR+ T cells. The TCR-Ts were then aliquoted and frozen.
3
Generation of P14 and OT-1 Immune Chimeras
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
P14 and OT-1 immune chimeras were generated by transferring 5 × 104 CD8+ T cells of either genotype into naive C57BL/6J mice, followed by i.n. infection with 500 PFU PR8-gp33 or 40 PFU PR8-ova (i.n.) 1 d later. R.A. Langlois made recombinant influenza viruses. Alternatively, mice were infected with 2 × 105 PFU Armstrong (i.p.) 1 d after cell transfer. For contemporaneous infections, mice were given 2.5 × 104 P14 and OT-1 cells and infected 1 d later. Where noted, CD8+ T cells were enriched using a negative-isolation kit (StemCell Technologies) supplemented with biotinylated Ly6C (1A8) antibody to remove circulating memory P14 cells. For in vitro peptide stimulations, cells were cultured for 4 h at 37°C in restimulation buffer (RPMI 1640 supplemented with 10% FBS, l -glutamine, sodium pyruvate, penicillin/streptomycin, Hepes, nonessential amino acids, and β-mercaptoethanol) containing 1× Golgi-plug (BD) and gp33 peptide (New England Peptide) at a final concentration of 0.2 µg/ml. For in vitro proliferation assays, cells were labeled with the cell tracking dye CellTracker Violet (Thermo Fisher Scientific) and cultured for 68 h at 37°C with 2 × 105 congenically distinct feeder cells in medium containing 0.66 µg/ml gp33 peptide.
4
Isolation and Characterization of HIV-specific CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD4+ T cells were isolated from thawed PBMCs using a negative isolation kit (StemCell, Cat# 19052), rested for 2 h at 37 °C, washed and stained for 60 min at room temperature with PE-labeled MHC-II tetramers loaded with DV16 peptide (DRFYKTLRAEQASQEV) for the DRB1*01:01 allele or YV18 peptide (YVDRFYKTLRAEQASQEV) for the DRB1*11:01 allele (NIH Tetramer Core Facility at Emory University, Atlanta, GA). These sequences encompass an immunodominant, HLA Class II promiscuous epitope in Gag [22] (link). Control tetramers loaded with an irrelevant peptide (CLIP: PVSKMRMATPLLMQA) or HIV-uninfected donors with the same HLA-DRB1 genotype served as negative controls. Tetramer+ CD4+ T cells were column enriched using anti-PE beads (Miltenyi, Cat# 130-048-801). Cells were stained for viability marker (Aquavivid, Thermofisher, Cat# L34957), CXCR5 (45 min, 37 °C), surface markers (30 min, 4 °C) and fixed with 2% PFA before acquisition at the flow cytometer (LSRIIB, BD).
5
Isolating CD8+ T-cells from Healthy Donors
6
Naïve CD4+ T Cell Treg Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Naïve CD4+ T cells were isolated from PBMCs using negative isolation kit (cat # 19555) purchased from Stem Cell Technologies (Vancouver, BC, Canada). The isolated naïve CD4+ T cells were then differentiated into Treg using ImmunoCult™ Human Treg Differentiation Supplement Catalog # 10977 which is a Serum-free culture supplement for the differentiation of human naïve CD4+ T cells into regulatory T cells (Tregs) based on the manufacturer’s recommendation. Briefly, CD4+ T cells (1 × 106 cell/mL) were cultured with ImmunoCult-XF T cell expansion medium and Immunocult Human CD3/CD28 T cell activator (25 µL/mL), both purchased from Stem Cell Technologies (Vancouver, BC, Canada). The cell density was adjusted to 1 × 106 cell/mL every 3–4 days as needed with the addition of fresh medium. ImmunoCult™ Human Treg Differentiation Supplement contains a human cytokine and small molecule formulated to promote the robust activation, expansion, and differentiation of peripheral blood-derived, naïve, CD4+ human T cells into regulatory T cells (Tregs).
7
Isolating CD8+ T-cells from Healthy Donors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human peripheral blood (buffy coats) from de-identified healthy donors were purchased (Oklahoma Blood Institute). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood via centrifugation with Lymphocyte Separation Media (Corning, 25–072-CV). Following PBMC isolation, CD8+ T-cells were isolated using a negative isolation kit from Stem Cell (17953). Cells were cryoed down in HI-FBS with 10% DMSO until further use.
8
Isolation and Activation of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ T cells and CD4+ T cells were isolated from spleens and peripheral lymph nodes, prepared into single cell suspensions and lysed for red blood cells (10x RBC lysis buffer, Biolegend). Negative isolation kits and positive isolation kits (CD11b+ cells) were purchased from StemCell Technologies and isolation was performed following manufacture’s procedures. T cells were activated and cultured as previously described (Jones et al., 2007 (link)) using plate-bound anti-CD3 and anti-CD28 antibodies.
Isolation of intratumoral CD11b+ cells was performed on tumors digested using the tumor preparation protocol (see below). CD11b+ cells were isolated from tumor single cell suspensions using positive isolation kits from StemCell Technologies, following the manufacturer’s protocol.
Macrophages were differentiated from bone marrow flushed using PBS, a 1mL syringe and a 25G needle. Bone marrow was collected, lysed for red blood cells (10x RBC lysis buffer, Biolegend), and plated on non-TC treated plates at 5x106 cells/10cm dish and 20ng/ml of M-CSF (Peprotech).
Cell culture of pancreatic ductal adenocarcinoma cells (PDACs) and ID8 cell lines were maintained in DMEM supplemented with 10% FBS and Pen/Strep.
Isolation of intratumoral CD11b+ cells was performed on tumors digested using the tumor preparation protocol (see below). CD11b+ cells were isolated from tumor single cell suspensions using positive isolation kits from StemCell Technologies, following the manufacturer’s protocol.
Macrophages were differentiated from bone marrow flushed using PBS, a 1mL syringe and a 25G needle. Bone marrow was collected, lysed for red blood cells (10x RBC lysis buffer, Biolegend), and plated on non-TC treated plates at 5x106 cells/10cm dish and 20ng/ml of M-CSF (Peprotech).
Cell culture of pancreatic ductal adenocarcinoma cells (PDACs) and ID8 cell lines were maintained in DMEM supplemented with 10% FBS and Pen/Strep.
9
Isolation and Characterization of Immune Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ and CD3+ cells were isolated from spleens and lymph nodes of mice using negative isolation kits (Stem Cell Technologies). Cell purity ranged between 95 and 99%, as tested by flow cytometry. MDSC were isolated from tumors previously digested with DNAse and Liberase (Roche USA, Branchburg, NJ) at 37°C for 1 hour, as previously described [25 ]. Purity ranged from 90 to 99% as measured by flow cytometry. Alternatively, MDSC were generated after culturing bone marrow cells for 4 days with 20 ng/ml G-CSF and GM-CSF [35 (link)], followed by sorting of GR-1+ cells. iMC were isolated from spleens of mice without tumors, as we described [25 ]. MDSC were depleted from T cells and MDSC co-cultures by selection of CD11b+ cells using magnetic-bead isolation (Stemcell technologies). T cell purity ranged from 96-100% as monitored by flow cytometry.
10
Isolation and Activation of T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ T cells and CD4+ T cells were isolated from spleens and peripheral lymph nodes, prepared into single-cell suspensions, and lysed for red blood cells (10× RBC lysis buffer, Biolegend). T cells were isolated by negative isolation kits (StemCell Technologies) and following manufacturer’s protocol. T cells were then activated and cultured as previously described48 (link) using plate-bound anti-CD3 and anti-CD28 antibodies in T cell medium (TCM) (IMDM, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 50 μM β-mercaptoethanol).
Mouse Pdx1-KrasG12D pancreatic cancer cell line was derived from the primary pancreatic tumours from the Pdx1-cre pancreatic cancer model. Cells were maintained in culture in DMEM, 10% FBS, and 1% penicillin-streptomycin. The EL4-OVA thymoma cell line was maintained in TCM with 0.4 mg ml−1 of G418 (Roche Diagnostic GmbH). All cells were incubated at 37 °C and 5% CO2 humidified incubators.
Mouse Pdx1-KrasG12D pancreatic cancer cell line was derived from the primary pancreatic tumours from the Pdx1-cre pancreatic cancer model. Cells were maintained in culture in DMEM, 10% FBS, and 1% penicillin-streptomycin. The EL4-OVA thymoma cell line was maintained in TCM with 0.4 mg ml−1 of G418 (Roche Diagnostic GmbH). All cells were incubated at 37 °C and 5% CO2 humidified incubators.
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!