PerCP Mouse Anti-Human CD4 (Cat# 347324), FITC Mouse Anti-Human CD25 (Cat#555431), PE Mouse anti-Human Foxp 3 (Cat# 560852), PE Mouse anti-Human IL-17A (Cat# 560486), Permeabilizing Solution 2 (Cat# 340973), Lysing Solution (10X) (Cat# 349202), and Protein Transport Inhibitor (Cat# 51-2092KZ) were purchased from BD Biosciences, USA. Red Blood Cell Lysis Buffer (Cat# 420301) was obtained from Biolegend, Inc. TRIzol solution (Cat# 15596026) was purchased from Ambon, USA. SYBR Green PCR kit (Cat# KM4101) and cDNA first strand synthesis kit (Cat# 639505) were provided by KAPA Biosystems, USA and Takara Bio, Japan respectively. DNase I (Cat# AM2295) was obtained from Fermentas, Lithuania. PCR primers were synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. All other reagents were of the highest quality available from commercial vendors.
Protein transport inhibitor
Manufactured by BD
Sourced in United States
The Protein Transport Inhibitor is a laboratory equipment used to disrupt the movement of proteins within a cell. It functions by interfering with the cellular mechanisms responsible for the transport and localization of proteins.
Automatically generated - may contain errors
Lab products found in correlation
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Cd3 apc cy7, by BioLegend (2 mentions)
Fixation and permeabilization solution kit, by BD (2 mentions)
Pe anti human ifn γ antibody, by BioLegend (2 mentions)
Beckman cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Permeabilizing solution 2, by BD (1 mentions)
Fitc mouse anti human cd25, by BD (1 mentions)
Pe mouse anti human foxp 3, by BD (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Roche (1 mentions)
Red blood cell lysis buffer, by BioLegend (1 mentions)
Pe mouse anti human il 17a, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti ifn γ, by BD (1 mentions)
Cd8 bv605, by BD (1 mentions)
Cd4 apc, by BioLegend (1 mentions)
Fitc anti cd107a, by BioLegend (1 mentions)
Anti il 17a bv421, by BioLegend (1 mentions)
Facs lsr 2, by BD (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
20 protocols using protein transport inhibitor
1
Comprehensive Immune Cell Profiling Protocol
2
Quantification of Murine Immune Cell Populations
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Peripheral blood mononuclear cells (PBMCs) were collected from 3 mice from each group at 14 days after the first immunization. Single cell suspensions (1 × 106 cells/mL) were prepared in PBS and stained with anti-mouse CD19 and CD40 antibodies (BD Biosciences, Franklin, TN, USA) to label the B cells and with anti-mouse CD3, CD4 and CD8 monoclonal antibodies (BD Biosciences, USA) to label the T cells. Splenocytes were isolated at two weeks after the second vaccination, and splenocyte suspensions (1 × 107 cells/ml) were stimulated with purified GP (10 μg/ml) associated with a protein transport inhibitor (BD Biosciences). At 6 h post-stimulation, surface staining was performed with anti-CD4 and anti-CD8 monoclonal antibodies for 0.5 h at 4 °C, and then the cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained with anti-IFN-γ and anti-IL-4 monoclonal antibodies (BD Biosciences), respectively, for 0.5 h at 4 °C. All labelled cells were washed twice with PBS, and 20,000 cells were analysed in a LSR-II flow cytometer (BD Biosciences).
3
Quantifying CAR-T Cell Cytotoxicity
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An E:T ratio of 1:1 was used when co‐culturing CD44v6 CAR‐T (or NT) cells with target cells in complete RPMI 1640 media containing anti‐CD107a‐FITC (Biolegend, USA) and protein transport inhibitor (BD Bioscience, USA) for 4 h. CD3‐APC/Cy7(Biolegend, USA), CD4‐APC (Biolegend, USA) and CD8‐BV605 (BD Bioscience, USA) were used to stain the harvested cells.
4
Analyzing T Cell Migration and Cytokine Production in Skin Constructs
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To analyze T cells that migrated into the skin constructs, skin constructs were incubated in 1 mg/ml collagenase type IV (Worthington) for 1 h, vortexing every 20 min. The cell suspension was filtered through a 70 μm cell strainer and washed with PBS with 2% FBS before staining. Isolated cells were stained using anti-CD3 FITC (OKT3, Biolegend), anti-CD25 PerCP 5.5 (M-A251, BD Bioscience), and anti-CD69 Pacific Blue (FN50, Biolegend).
To analyze intracellular cytokines, the cells were stimulated by adding cell stimulation cocktail (eBioscience) for 1 h in addition to protein transport inhibitor (BD Biosciences) for 4 h. Cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions and were stained using anti-IFNγ PE (4 S.B3, BD Biosciences), anti-IL-17A BV421 (BL168, Biolegend). All stained cells were analyzed on a FACS LSR II (BD Biosciences) and FCS express 6.0 (De Novo Software).
To analyze intracellular cytokines, the cells were stimulated by adding cell stimulation cocktail (eBioscience) for 1 h in addition to protein transport inhibitor (BD Biosciences) for 4 h. Cells were fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions and were stained using anti-IFNγ PE (4 S.B3, BD Biosciences), anti-IL-17A BV421 (BL168, Biolegend). All stained cells were analyzed on a FACS LSR II (BD Biosciences) and FCS express 6.0 (De Novo Software).
5
ADCC Assay for Antibody Function
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The ADCC assay was performed using a previously published method [23 (link)]. Briefly, PBMCs containing NK cells were co-cultured with interleukin-2 overnight. CSP-coated beads were opsonized with a pool of sera selected from the RTS,S vaccination group. The opsonized beads were co-cultured with the primed PBMC and anti-CD107a-AF647 for 1 h followed by the addition of brefeldin A (Sigma-Aldrich) and protein transport inhibitor (BD Bioscience) and further co-cultured for 3 h. After co-culture, the cells were stained with anti-CD3-APC-H7 (SK7, BD Bioscience), anti-CD56-PE-Cy7 (B159, BD Bioscience) and anti-CD16-BV421 (3G8, BD Bioscience). NK cells were defined as CD3−, CD56+, and CD16+and the level of ADCC was quantified as the percentage of NK cells that were positive for CD107a staining by flow cytometry (LSR Fortessa X-20, BD Bioscience; gating strategy is shown in a previous publication [23 (link)]).
6
Murine Lymphocyte Immunophenotyping by Flow Cytometry
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Mouse peripheral blood was collected and prepared into lymph node single cell suspension for flow cytometry assay. Cells were harvested and stained with anti-CD4 antibodies (all from BD Biosciences, USA). FoxP3 staining was performed using Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kit (eBioscience, USA) according to manufacturer's instruction. Then, cells were stained with anti-FoxP3 antibody (eBioscience). Regarding intracellular cytokine staining, spleen cells were cultured in media containing PMA (50 ng/ml) and Ionomycin (1 μg/ml) plus protein transport inhibitor (BD Biosciences) for 5 hours. Cells were then fixed, permeabilized, and stained with anti-IL-17A (all from eBioscience, USA). Finally, cells were analyzed using a flow cytometer (FACSCalibur, BD Biosciences). Fluorescence minus one (FMO) samples and unstained samples were used as internal controls to set positive and negative gating. Cell gating and data collection were performed in a blinded manner.
7
Cytokine Profiling of Immunized Mice
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Splenocytes were collected from 3 mice from each group at 14 days after immunization and were stimulated with inactivated HuNPB3 (10 μg/mL) with a protein transport inhibitor (BD Biosciences, Franklin, TN, USA). At 6 h post-stimulation, single cell suspensions were stained with anti-CD4 and anti-CD8 monoclonal antibodies (BD Biosciences, Franklin, TN, USA) for 30 min at 4 °C, as well as anti-IFN-γ and anti-IL-4 monoclonal antibodies (BD Biosciences, Franklin, TN, USA) for 30 min at 4 °C. All labeled cells were washed twice with PBS and 20,000 cells were analyzed in a LSR-II flow cytometer (BD Bioscience, Franklin, TN, USA).
8
Intracellular IFN-β Detection in MDDCs and THP-1
9
PBMC Cytokine and Checkpoint Profiling
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We cultured PBMCs in 48-well plates and added Phorbol 12-myristate 13-acetate (PMA) (50 ng/ml, TOCRIS, USA) plus ionomycin calcium salt (1 μM/ml, Sigma-Aldrich, USA) for 16 h. We added protein transport inhibitor (BD Bioscience, USA) to the co-culture 12 h before we stopped the stimulation. After coculturing, PBMCs were stained for CD4, CD8, interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and TIM3 expression. The cytokine and immune checkpoint expressions were measured by flow cytometry and were analyzed in BD FACSuite V software (BD, Biosciences, USA).
10
Splenocyte Stimulation and Flow Cytometry
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Individual spleens were harvested at d42 into Hank’s balanced salt solution at room temperature (RT) without calcium or magnesium (HBSS -/-) (Wisent, St. Bruno, QC), and processed as previously described [18 (link)]. Fresh splenocytes were seeded in 96-well U-bottom plates (BD Falcon, Mississauga, ON) in 200 μL per well (1x106 cells/well). Duplicate cultures were stimulated with cRPMI alone (unstimulated), H1-VLP (2.5 μg/mL HA) or PMA+ ionomycin (each 1 mg/mL) (Sigma, St. Louis, MO) for 18 hours at 37°C at 5% CO2. After 13 hours, protein transport inhibitor was prepared according to manufacturer’s instructions (BD Science, San Jose, CA) and added to samples (20 μL/well). Five hours later, plates were spun (320xg, 8 minutes at 4°C) and cells were processed for flow cytometry as described below. In our hands, we see no significant differences in T cell responses after splenocyte re-stimulation ex vivo with inactivated virions, VLPs or recombinant HA.
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