Cells were washed and incubated with near-infrared fixable live/dead stain (Life Technologies), as per the manufacturer’s instructions. Cells were washed before incubation with human Fc Block (BD Pharmingen), and surface staining was performed with a lineage cocktail of the following antibodies (clones in parentheses) purchased from BioLegend: CD3 (HIT3a), CD4 (OKT4), CD8 (HIT8a), CD19 (HIB19), CD20 (2H7), CD34 (561), FcER1 (AER-37), CD45 (HI30), CX3CR1 (2A9-1), CCR2 (K036C2), CD36 (5–271), CD11b (M1/70), CD11c (3.9), CD16 (B73.1), CD163 (GHI/61), CD14 (M5E2), CD206 (15–2), CD86 (IT2.2), CSFR1 (9-4D2-1E4; BD Biosciences), and HLA-DR (G46-6). Total macrophage populations were identified as auto-fluorescent, CD11c+ cells. Surface staining was followed by fixation and then permeabilization to allow for intracellular or intranuclear staining. Labeled cells were acquired on a fluorescence-activated cell sorter (LSR Fortessa II; BD Bioscience) and further analyzed using FlowJo (Tree Star).
Near infrared fixable live dead stain
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Near-Infrared Fixable Live/Dead stain is a fluorescent dye that can be used to distinguish between live and dead cells. It binds to cellular proteins, and its fluorescence emission can be detected using flow cytometry or microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (4 mentions)
Cytofix cytoperm, by BD (4 mentions)
Cd8α fitc 76 2 11, by BD (4 mentions)
Lsrfortessa, by BD (3 mentions)
Cd4 clone 74 12 4 percpcy5, by BD (3 mentions)
Ifn γ pe p2g10, by BD (3 mentions)
Cross reactive anti human tnf α af650 mab11, by BioLegend (3 mentions)
Human fc block, by BD (2 mentions)
Cd3ε af647 bb23 8e6 8c8, by BD (2 mentions)
Lsr fortessa 2, by BD (1 mentions)
Aria 3, by BD (1 mentions)
Macs system, by Miltenyi Biotec (1 mentions)
Lsr fortessa instrument, by BD (1 mentions)
Anti mouse ig fitc, by Southern Biotech (1 mentions)
Goat anti mouse igg bv421, by BioLegend (1 mentions)
Streptavidin af647, by BioLegend (1 mentions)
Facs lsrii, by BD (1 mentions)
Phospho p53 ser15 antibody, by Cell Signaling Technology (1 mentions)
Anti cd8 apc h7, by BD (1 mentions)
Anti pd 1 bv711, by BioLegend (1 mentions)
9 protocols using near infrared fixable live dead stain
1
Comprehensive Immune Cell Phenotyping
2
Isolation and Characterization of CD206+ Alveolar Macrophages
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CD206+ AMs were enriched from donor BAL using the magnetic-based MACS® system (Miltenyi Biotech, Germany) and fluorescent activated cells sorting (FACS) as outlined previously 3, (link)20, (link)21 . Briefly, for MACS-based enrichment, BAL cells (1 × 10 7 ) were incubated with human Fc-block (BD Biosciences, USA) and human CD206 APC-Cy7 (clone 15-2, BioLegend, USA). CD206+ cells were enriched in MACS LS magnetic separation column and MidiMACS TM magnet. Cell counts were determined using a haemocytometer and trypan blue live/dead exclusion.
For FACS-based enrichment BAL cells were washed and incubated with nearinfrared fixable live/dead stain (Life Technologies Inc.) as per the manufacturer's instructions. After incubation with human Fc block (BD Pharmingen, Inc.), surface staining was performed with the following antibodies (fluorophore followed by clone in parentheses); CD45 (PE-Texas Red, H130), CD3 (FITC, OKT), TCR-β (BV421, IP26), CD206 (PercpCy5.5, 15.2). Cell sorting was carried out on Aria III (BD Biosciences) and AMs defined as live, CD45 + CD3 -TCR -CD206 + cells.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.04.410191 doi: bioRxiv preprint
For FACS-based enrichment BAL cells were washed and incubated with nearinfrared fixable live/dead stain (Life Technologies Inc.) as per the manufacturer's instructions. After incubation with human Fc block (BD Pharmingen, Inc.), surface staining was performed with the following antibodies (fluorophore followed by clone in parentheses); CD45 (PE-Texas Red, H130), CD3 (FITC, OKT), TCR-β (BV421, IP26), CD206 (PercpCy5.5, 15.2). Cell sorting was carried out on Aria III (BD Biosciences) and AMs defined as live, CD45 + CD3 -TCR -CD206 + cells.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted December 4, 2020. ; https://doi.org/10.1101/2020.12.04.410191 doi: bioRxiv preprint
3
Cytokine Analysis of Influenza-Stimulated PBMCs
4
Profiling Influenza-Specific T-Cell Responses
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Cryopreserved mononuclear cells from blood, TBLN and BAL were thawed and stimulated for 12 h at 37°C with live MDCK-grown wt H1N1 or wt H3N2 (MOI = 1) or media control. GolgiPlug (BD Biosciences, UK) was added according to the manufacturer's instructions for a further 5 h before intracellular cytokine staining (ICS). Cells were stained for surface markers with CD3ε-PeCy7 BB23-8E6-8C8, CD4 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 (BD Biosciences, UK) and Near-Infrared Fixable Live/Dead stain (Invitrogen, UK). Cells were fixed and permeabilized using Cytofix Cytoperm (BD Biosciences, UK) before intracellular staining with IFNγ AF647 P2G10 (BD Biosciences, UK) and cross-reactive anti-human TNFα-BV650 Mab11 (BioLegend, UK). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa (BD Biosciences). Data was analyzed by Boolean gating using FlowJo v10 (Treestar).
5
Cytokine profiling of influenza-specific T cells
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Cryopreserved mononuclear cells from blood, TBLN, BAL, spleen, and lung were thawed and stimulated for 12 h at 37°C with either 1 × 106 PFU live MDCK-grown strain 1353/09pdmH1N1 or 1 × 106 TCID50 H3N2 S-FLU or MDCK mock supernatant as control. GolgiPlug (BD Biosciences) was added for a further 4 h before intracellular cytokine staining. Cells were stained for surface markers with CD3ε-AF647 BB23-8E6-8C8, CD4 clone 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 (BD Biosciences), and Near-Infrared Fixable LIVE/DEAD stain (Invitrogen). Cells were permeabilized using Cytofix/Cytoperm (BD Biosciences) before intracellular staining with IFN-γ PE P2G10 (BD Biosciences) and cross-reactive anti-human TNF-α-AF650 Mab11 (BioLegend). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa (BD Biosciences). Data were analyzed using FlowJo v10 (Tree Star).
6
Cryopreserved Immune Cell Cytokine Response
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Cryopreserved PBMC and cells from TBLN and BAL were thawed and stimulated for 12 h at 37 °C with live MDCK-grown virus strain A/Sw/Eng/1353/09 (MOI 6 for BAL and PBMC, MOI 0.6 for TBLN) or MDCK mock supernatant as control. GolgiPlug (BD Biosciences) was added according to the manufacturer’s instructions for a further 5 h before intracellular cytokine staining. Cells were stained for surface markers with CD3ε-PeCy5 PPT3 (AbCam), biotinylated CD4 clone MIL17 (in-house), with secondary streptavidin-APC (Southern Biotec), CD8α-FITC MIL12 (AbD Serotec) and Near-Infrared Fixable Live/Dead stain (Invitrogen). Cells were permeabilized using Cytofix Cytoperm (BD Biosciences) as per the manufacturer’s instructions before intracellular staining (ICS) with IFN-γ PE P2G10 (AbD Serotec). Samples were fixed in 1% paraformaldehyde before analysis using an LSR Fortessa instrument (BD Biosciences).
Data was analysed using FlowJo v10 (Treestar), fluorescence of control samples stained with one primary antibody omitted were used to set gates. Samples were batch gated on lymphocytes based on SSCA/FSCA, followed by single cells on SSCH/SSCA. Live CD3 positive cells were analysed for expression of CD4 and CD8α. Boolean gating was used to determine the levels of IFN-γ in CD8α high, CD4CD8α double positive and CD4 T cell subsets.
Data was analysed using FlowJo v10 (Treestar), fluorescence of control samples stained with one primary antibody omitted were used to set gates. Samples were batch gated on lymphocytes based on SSCA/FSCA, followed by single cells on SSCH/SSCA. Live CD3 positive cells were analysed for expression of CD4 and CD8α. Boolean gating was used to determine the levels of IFN-γ in CD8α high, CD4CD8α double positive and CD4 T cell subsets.
7
Quantifying Intravascular and Tissue-Resident T Cells
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Before sacrifice, three animals from each group were infused i.v. with 10 ml of 3.24 mg/ml purified CD3 Ab (clone PPT3) and sacrificed 10 min later. Lymphocytes were isolated and stained ex vivo with anti-mouse Ig-FITC (SouthernBiotech), which labels the circulating intravascular cells. The cells are washed, and normal mouse serum is then added to block any remaining binding capacity of the anti–Ig-FITC. The cells are then washed again and incubated with CD3 Ab labeled with PeCy5 (Abcam). This will bind unsaturated sites of the circulating cells, which are therefore double labeled, as well as all the sites on the TRM that are not accessible to the CD3 Ab, given i.v. TRM is therefore single labeled with PeCy5.
To allow intracytoplasmic staining of TRM, the i.v. CD3 was detected with goat anti-mouse IgG BV421 (BioLegend) and blocked using normal mouse serum as above. Surface markers used were CD3ε-biotin PPT3 (Abcam), CD4 clone 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 (all BD Biosciences), and Near-Infrared Fixable LIVE/DEAD stain (Invitrogen). Biotinylated CD3 was visualized with a streptavidin AF647 (BioLegend). Cells were permeabilized using Cytofix/Cytoperm before intracellular staining with IFN-γ PE P2G10 (BD Biosciences) and cross-reactive anti-human TNF-α-AF650 Mab11 (BioLegend). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa.
To allow intracytoplasmic staining of TRM, the i.v. CD3 was detected with goat anti-mouse IgG BV421 (BioLegend) and blocked using normal mouse serum as above. Surface markers used were CD3ε-biotin PPT3 (Abcam), CD4 clone 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 (all BD Biosciences), and Near-Infrared Fixable LIVE/DEAD stain (Invitrogen). Biotinylated CD3 was visualized with a streptavidin AF647 (BioLegend). Cells were permeabilized using Cytofix/Cytoperm before intracellular staining with IFN-γ PE P2G10 (BD Biosciences) and cross-reactive anti-human TNF-α-AF650 Mab11 (BioLegend). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa.
8
Cell Viability Assessment by Flow Cytometry
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Infected cells were washed twice in PBS and lifted with 2X trypsin (Life Technologies), before quenching in DMEM 10%FBS. The cell pellet was washed in PBS before staining with a fixable live/dead near infrared stain (Thermofisher) at 1/2000 in PBS for 20min on ice. PBS was added to quench the reaction before the suspension was centrifuged at 1250 rpm for 5 min at 4°C. The cell pellet was then fixed in 3% paraformaldehyde in PBS, for 20min on ice. PBS was added to quench the reaction before centrifugation at 1250 rpm for 5min at 4°C. The cells were resuspended in PBS 1%BSA 2.5mM EDTA and analyzed on a BD LSR-II FACS. Results were analyzed using FlowJo V.10.1 software.
9
Comprehensive Immunophenotyping of CAR T-Cells
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For measurement of surface markers, cells were stained with either fixable live/dead violet or near-infrared stain (Thermo) in PBS depending on the panel, followed by surface staining in PBS containing 2.5% heat-inactivated FBS (Seradigm). CD19 CAR staining was performed using an AF647 or APC conjugated anti-idiotype antibody directed at the FMC63 scFv. CD8 staining was performed using anti-CD8 APC-H7 (BD) and finally PD1 staining was performed using anti-PD1 BV711 (BioLegend). Measurement of intracellular markers was performed by first staining cells with fixable live/dead near-infrared stain (Thermo). After washing, cells were fixed using 1.5% paraformaldehyde at room temperature for 10 min. Washed cells were then resuspended in cold methanol and stained on ice for 15 min. Cells were then either stored at −80 °C for temporary storage to enable batch analysis or were immediately washed and resuspended in PBS with 2.5% FBS (Seradigm) prior to intracellular staining. Phosphorylated p53 serine 15 was stained using phospho-p53 Ser15 antibody conjugated to either PE or AF488 depending on the panel (Cell Signaling Technology). All data were collected by a Fortessa LSR II cytometer (BD Biosciences) equipped with FACSDiva software. Analysis was performed using FlowJo version 10 (BD Biosciences).
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