ICS staining and flow cytometry were performed as described in detail (61 (link)), except differing in the peptide target. In brief, PBMC were incubated with pooled overlapping 15-mer peptides spanning spike (67 (link)) containing 1µg/ml each peptide, with brefeldin A (catalog #00-4506-51, eBioscience, San Diego, CA) and monensin (#00-4505-51, eBioscience, San Diego, CA), followed by surface staining with CD3-Super Bright 436, CD8-Super Bright 600, CD4 PE-Cy7, and Fixable Aqua viability dye (catalog #62-0037-42/eBioscience/San Diego/CA, #63-0088-42/eBioscience/San Diego/CA, #25-0049-42/San Diego/CA, and #L34957/Invitrogen/Waltham/MA respectively), permeabilization (catalog #00-5523-00, eBioscience, San Diego, CA), and intracellular cytokine staining for IFN-γ-FITC, IL-2-PerCP-Cy5.5, IL-4-PE, and IL-10-APC (catalog #506504/Biolegend/San Diego/CA, #500322/Biolegend/San Diego/CA, # 130-091-647/Miltenyi Biotec/Bergisch Gladbach/Germany, and #506807/Biolegend/San Diego/CA respectively) followed by flow cytometric analysis.
Fixable viability dye aqua
Manufactured by Thermo Fisher Scientific
Fixable viability dye aqua is a fluorescent dye used to detect and quantify dead cells in flow cytometry applications. It binds to proteins in cells with compromised membranes, providing a measure of cellular viability.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 pe cy7, by Thermo Fisher Scientific (2 mentions)
Brefeldin a, by Thermo Fisher Scientific (2 mentions)
Monensin, by Thermo Fisher Scientific (2 mentions)
Cd8 super bright 600, by Thermo Fisher Scientific (2 mentions)
Cd3 brilliant violet 785 clone okt3, by BioLegend (2 mentions)
Cd8 bv605 clone sk1, by BioLegend (2 mentions)
Mhc 1 pacblue clone w6 32, by BioLegend (2 mentions)
Gag kc57 fitc, by Beckman Coulter (2 mentions)
Cd4 apc r700 clone rpa t4, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Lsr 2, by BD (2 mentions)
Cd62l, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Merck Group (2 mentions)
E50 2440, by BD (2 mentions)
Cd3 super bright 436, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Collagenase d, by Roche (1 mentions)
Ra3 6b2, by BD (1 mentions)
Collagenase, by Roche (1 mentions)
Facscanto 2 cytofluorometer, by BD (1 mentions)
11 protocols using fixable viability dye aqua
1
Spike Protein-Specific T-cell Assay
2
Spike-specific T-cell Immune Response Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ICS staining and flow cytometry were performed as described in detail [12 (link)], except differing in the antigenic target. In brief, PBMCs were incubated with a pool of overlapping 15-mer peptides spanning spike [13 ] at a final concentration of 1 µg/ml of each individual peptide, with brefeldin A (#00-4506-51, eBioscience, San Diego, CA) and monensin (#00-4505-51, eBioscience, San Diego, CA), followed by surface staining CD3-Super Bright 436, CD8-Super Bright 600, CD4 PE-Cy7, and Fixable Aqua viability dye (#62-0037-42, eBioscience, San Diego/CA; #63-0088-42, eBioscience, San Diego/CA; #25-0049-42, San Diego, CA; and #L34957, Invitrogen, Waltham, MA respectively), permeabilization (#00-5523-00, eBioscience, San Diego, CA), and intracellular cytokine staining for interferon (IFN)-ɣ-FITC, IL-2-PerCP-Cy5.5, IL-4-PE, and IL-10-APC (#506504 Biolegend, San Diego, CA; #500322, Biolegend, San Diego, CA; # 130-091-647, Miltenyi Biotec, Bergisch Gladbach, Germany; and #506807, Biolegend, San Diego, CA respectively) for flow cytometric analysis.
3
Measurement of Antigen Presentation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure antigen presentation, 293T cells were transduced with a retrovirus vector (LMNi-bsd) expressing HLA A11 or an empty vector control. The cells were subsequently transduced with empty SCRPSY or SCRPSY expressing an ORF7a protein at an MOI of 1. After selection in puromycin for 24 h, cells were transfected with an HIV-1 Gag expression plasmid. these “target” cells were washed to remove the selection media, plated in 96-well plates and then cocultured with a Gag-AK11-specific CD8+ T cell clone in RPMI medium 1640 supplemented with 10% FBS, 2 mM l-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, and 50 units/mL IL-2 (NCI BRB Preclinical Biologics Repository) at 37 °C 5% CO2. After 16 h, cells were pelleted, and supernatants were harvested for IFN-γ ELISA. Cells were then washed in 2% FBS phosphate-buffered saline + 2 mM EDTA and surface stained with fluorochrome-conjugated antibodies to CD3-Brilliant Violet 785 clone OKT3, CD8-BV605 clone SK1, MHC-I- PacBlue clone W6/32, (all from BioLegend), CD4-APC R700 clone RPA-T4, (BD), Gag KC57-FITC (Beckman Coulter), and Fixable Aqua Viability Dye (Invitrogen). Counting beads were also added. Samples were analyzed by flow cytometry, and data analysis was performed with FlowJo (version 10) software.
4
Comprehensive Immune Profiling of Tumor Samples
5
Antigen Presentation Evaluation by Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To measure antigen presentation, 293T cells were transduced with a retrovirus vector (LMNibsd) expressing HLA A11, or an empty vector control. The cells were subsequently transduced with empty SCRPSY or SCRPSY expressing an ORF7a protein at an MOI of 1. After selection in puromycin for 24 hours cells were transfected with an HIV-1 Gag expression plasmid. these ‘target’ cells were washed to remove the selection media, plated in 96 well plates and then cocultured with a Gag-AK11-specific CD8+ T-cell clone in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 units/ml IL-2 (NCI BRB Preclinical Biologics Repository) at 37°C 5% CO2. After 16 hours, cells were pelleted and supernatants were harvested for IFN-γ ELISA. Cells were then washed in 2% FBS phosphate-buffered saline + 2mM EDTA and surface stained with fluorochrome-conjugated antibodies to CD3-Brilliant Violet 785 clone OKT3, CD8-BV605 clone SK1, MHC-I- PacBlue clone W6/32, (all from BioLegend), CD4-APC R700 clone RPA-T4, (BD), Gag KC57-FITC (Beckman Coulter) and Fixable Aqua Viability Dye (Invitrogen). Counting beads were also added. Samples were analyzed by flow cytometry, and data analysis was performed with FlowJo, version 10, software.
6
Multicolor Flow Cytometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used were CD4, CD8, CD44, and CD62L (eBioscience Affymetrix). Cells were acquired on a BD FACSCalibur, and data were analyzed in FlowJo (Tree Star). SVF was stained for Fixable Viability Dye Aqua (Life Technologies), B220, MHCII, F4/80, and CD11b. Data were acquired on a BD LSR II and analyzed in FlowJo.
7
Multicolor Flow Cytometry Analysis
8
Lung Cell Isolation and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Harvested organs were
cut into small pieces and incubated for 1 h at 37 °C with a mix
of DNase I (100 μg/mL, Sigma-Aldrich) and collagenase D (400
U/mL, Roche). Lung cells were washed and filtered before being incubated
with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC)
in 200 μL of PBS, 0.2% BSA, and 0.02% NaN3 (FACS
buffer) for 20 min at 4 °C to prevent antibody binding to Fc
receptors. Various fluorescent monoclonal antibody (mAb) combinations
in FACS buffer were used to stain (3–5) × 106 cells. Acquisitions were done on a FACScanto II cytofluorometer
(Becton Dickinson) with the following mAbs: fluorescein (FITC)-coupled
anti-CD3 (145-2C11, BD Biosciences), FITC-coupled anti-CD11c (HL3,
ThermoFisher), FITC-coupled anti LY6G (1A8, BD Biosciences), phycoerythrine
(PE)-coupled anti-SiglecF (E50-2440, BD Biosciences), PE-coupled anti-MHCII
(M5, BD Biosciences), PE-coupled anti CD11b (M1/70, BD Biosciences),
allophycocyanin (APC)-coupled anti-F4/80 (BM8, BD Biosciences), APC-coupled
anti-B220 (RA3-6B2, BD Biosciences), APC-coupled anti-CD11c (HL3,
BD Biosciences), Brillant violet 421 (BV421)-coupled anti SiglecF
(E50-2440, BD Biosciences), BV421-coupled anti-MHCII (M5, BD Biosciences).
Fixable viability dye aqua (ThermoFisher) was used to gate viable
cells.
cut into small pieces and incubated for 1 h at 37 °C with a mix
of DNase I (100 μg/mL, Sigma-Aldrich) and collagenase D (400
U/mL, Roche). Lung cells were washed and filtered before being incubated
with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC)
in 200 μL of PBS, 0.2% BSA, and 0.02% NaN3 (FACS
buffer) for 20 min at 4 °C to prevent antibody binding to Fc
receptors. Various fluorescent monoclonal antibody (mAb) combinations
in FACS buffer were used to stain (3–5) × 106 cells. Acquisitions were done on a FACScanto II cytofluorometer
(Becton Dickinson) with the following mAbs: fluorescein (FITC)-coupled
anti-CD3 (145-2C11, BD Biosciences), FITC-coupled anti-CD11c (HL3,
ThermoFisher), FITC-coupled anti LY6G (1A8, BD Biosciences), phycoerythrine
(PE)-coupled anti-SiglecF (E50-2440, BD Biosciences), PE-coupled anti-MHCII
(M5, BD Biosciences), PE-coupled anti CD11b (M1/70, BD Biosciences),
allophycocyanin (APC)-coupled anti-F4/80 (BM8, BD Biosciences), APC-coupled
anti-B220 (RA3-6B2, BD Biosciences), APC-coupled anti-CD11c (HL3,
BD Biosciences), Brillant violet 421 (BV421)-coupled anti SiglecF
(E50-2440, BD Biosciences), BV421-coupled anti-MHCII (M5, BD Biosciences).
Fixable viability dye aqua (ThermoFisher) was used to gate viable
cells.
9
Characterizing Lung Cell Populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A described previously [53 (link)], lungs were harvested, cut into small pieces and incubated for 1 hour at 37°C with a mix of DNAse I (100 μg/ml, Sigma-Aldrich) and collagenase (400 U/ml 1.6 mg/ml, Roche). Lung cells were washed and filtered through a 100 μM filter before being incubated with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC) in 200 μL PBS 0.2% BSA 0.02% NaN3 (FACS buffer) for 20 minutes at 4°C to prevent antibody binding on the Fc receptor. Various fluorescent mAb combinations in FACS buffer were used to determine cell populations (Table 1 ). Acquisitions were done on FACScanto II cytofluorometer (Becton Dickinson) with the following mAbs from BD Biosciences: Fluorescein (FITC)-coupled HL3 (anti-CD11c), FITC-coupled 145-2C11 (anti-CD3), APC-coupled RB6-8C5 (anti-GR1), phycoerythrine (PE)-coupled RM4-5 (anti-CD4), PE-coupled E50-2440 (anti-SIGLEC-F), APC-coupled BM8 (anti-F4/80). APC-eF780-coupled M1/70 (antiCD11b) were purchased from eBiosciences and fixable viability dye Aqua (ThermoFisher) was used to gate viable cells. Gating strategies are summarized in S3 Fig
10
Intracellular Transcription Factor Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to intracellular transcription factor staining, dead cells were labeled with fixable viability dye aqua (Thermofisher). Cells were fixed and stained at room temperature using the FoxP3/Transcription Factor Staining kit (eBioscience) according to the manufacturer's recommendations with fixing and staining times of 1 h each. The following antibodies were used: T-Bet-PE (4B10, BioLegend) and FoxP3-eF450 (FJK-16s, eBioscience).
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!