For intracellular cytokine staining (ICS), cells were stimulated with S269–278 peptide or vaccinia virus peptide F226–34 for 2 h. Then, brefeldin A was added and cells were further stimulated for 4 h. After stimulation, cells were stained with anti–CD3-PE, anti-CD4 Brilliant Violet 421, anti-CD8α Alexa Fluor 488, and purified CD16/CD32. Cells were then washed, fixed, permeabilized with Perm Wash buffer, and stained intracellularly anti–IFN-γ plus anti–TNF-α. Data were acquired by a MACSQuant flow cytometer and analyzed using FlowJo software.
Macsquant flow cytometer
Manufactured by FlowJo
Sourced in United Kingdom
The MACSQuant flow cytometer is a multi-parameter analytical instrument designed for cell analysis and sorting. It is capable of rapidly measuring and analyzing the physical and fluorescent characteristics of cells or particles suspended in a fluid stream.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide solution, by Miltenyi Biotec (2 mentions)
7 aad, by BD (1 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Erythrocyte lysis buffer, by Qiagen (1 mentions)
Percp anti cd4 rm4 5, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Southern Biotech (1 mentions)
Fcr block, by Miltenyi Biotec (1 mentions)
Fty720, by Merck Group (1 mentions)
Anti cd4 vioblue, by Miltenyi Biotec (1 mentions)
Foxp3 transcription factor fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions)
Live dead fixable viability dye, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Anti calr antibody, by Abcam (1 mentions)
Macsquant, by FlowJo (1 mentions)
10 protocols using macsquant flow cytometer
1
Intracellular Cytokine Staining Workflow
2
Evaluating Chemical Cytotoxicity in CEFs
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To identify nontoxic concentrations of the chemicals, mock-infected and MDV-infected CEFs were exposed to the chemicals or vehicles, and cell morphology and adherence/confluence were monitored by light microscopy at different time points posttreatment. Moreover, CEFs were trypsinized, stained with 7-AAD (BD Bioscience, Oxford, UK), and acquired using a MACSQuant flow cytometer and FlowJo software for analysis of the data. Nontoxic concentrations of the inhibitors and chemicals were selected based on flow cytometry data and confluence.
3
Flow Cytometric Analysis of Dendritic Cell Phagocytosis and Maturation
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After 4 h, cells were detached with a cell scraper, and cell suspensions were transferred to 96-well plates. After washing with 150 μl/well of cold (4 °C) PBS, cells were stained with 50 μl/well of anti-CD11c antibody coupled to the PE-Vio770 fluorophore diluted in PBS/1% BSA. After 30 min of incubation on ice, cells were washed and fixed with 3.7% formaldehyde diluted in PBS. Finally, cells were analyzed by means of a MACSQuant flow cytometer, and data analyzed with FlowJo software. Percentage of phagocytized cells equaled to the percentage of CMFDA+ cells within CD11c+ BMDCs (phagocytosis assay) [66 (link)]. BMDCs maturation assay was performed after 24 h of co-culture. First, cells were detached with a cell scraper and transferred to 96-well plates. After washing with 150 μl/well of cold PBS, cells were incubated with 50 μl/well of the LIVE/DEAD fixable violet dead cell stain kit (Invitrogen) for 15 min on ice. After 2 additional washings, cells were stained with fluorescent anti-CD11c, -CD86, and -Ia/Ie antibodies for 30 min on ice. Finally, cells were fixed with 3.7% formaldehyde (diluted in PBS) and fluorescence intensity of single cells was quantified using a MACSQuant flow cytometer. Data were analyzed with FlowJo software. Fold increase of expression was calculated using the median of fluorescence intensity of each marker [67 (link)].
4
Multiparametric Flow Cytometry Analysis of Immune Cells
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Organs were freshly harvested and meshed on a 70 μm filter. Thereafter, samples were lysed with erythrocyte lysis buffer (Qiagen GmbH), washed twice with FACS buffer (3% BSA in 0.01 M PBS pH 7.2) and blocked with FcR block (Miltenyi Biotec) prior to staining. Subsequently, cells were incubated for 30 min at 4°C with the following antibodies: 3 μg/mL PE-Vio770 conjugated anti-CD3 (REA641, Miltenyi Biotec), 2 μg/mL PE anti-CD8a (53-6.7, eBioscience), 2 μg/mL PerCP anti-CD4 (RM4-5, BD Biosciences), and 3 μg/mL APC-Vio770 anti-CD19 (6D5, Miltenyi Biotec) antibodies. 1:1,000 (v/v) DAPI (SouthernBiotech) was used for discrimination of dead cells. Samples were acquired on a MACSQuant flow cytometer and analyzed with FlowJo software V10.
5
FTY720 Modulation of Tick-Host Immunity
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C57BL/6J (WT) mice were injected intraperitoneally (i.p.) with FTY720 (Sigma-Aldrich), 1 mg/kg in 100 μL sterile water on days −1 and on the morning of day 0 prior to tick placement.
Ticks were microinjected with vamp33 si or vamp33 sc. Ticks fed on FTY720-injected and PBS-injected mice for 3 days. Mice were euthanized on the third day. Tick weights were measured. In all, 10 mm skin punch biopsies and draining lymph nodes were obtained from euthanized mice. FTY720 treatment was confirmed through FACS measurement of lymphocytes in the skin and draining lymph nodes. Skin and lymph node samples were labeled using APC-Cy7 anti-CD45 (Miltenyi Biotec 130-110-662; 3 ng/μl), VioBlue anti-CD4 (Miltenyi Biotec 130-118-568; 3 ng/μl), PerCP-Vio700 anti-TCRγ/δ (Miltenyi Biotec 130-117-665; 3 ng/μl), and PECy7 CD3 (Miltenyi Biotec 130-116-530; 3 ng/μl). Live and dead cells were discriminated using propidium iodide solution (Miltenyi Biotec). Cell populations were measured with a MACSQuant flow cytometer and analysis was performed using FlowJo software v 10.6.1.
Ticks were microinjected with vamp33 si or vamp33 sc. Ticks fed on FTY720-injected and PBS-injected mice for 3 days. Mice were euthanized on the third day. Tick weights were measured. In all, 10 mm skin punch biopsies and draining lymph nodes were obtained from euthanized mice. FTY720 treatment was confirmed through FACS measurement of lymphocytes in the skin and draining lymph nodes. Skin and lymph node samples were labeled using APC-Cy7 anti-CD45 (Miltenyi Biotec 130-110-662; 3 ng/μl), VioBlue anti-CD4 (Miltenyi Biotec 130-118-568; 3 ng/μl), PerCP-Vio700 anti-TCRγ/δ (Miltenyi Biotec 130-117-665; 3 ng/μl), and PECy7 CD3 (Miltenyi Biotec 130-116-530; 3 ng/μl). Live and dead cells were discriminated using propidium iodide solution (Miltenyi Biotec). Cell populations were measured with a MACSQuant flow cytometer and analysis was performed using FlowJo software v 10.6.1.
6
Macrophage Uptake of Phosphatidylcholine
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Analysis of PC uptake by primary human macrophages was conducted using flow cytometry 3 h after PC supplementation (day 7). Cells were harvested as above, washed, and immediately analyzed for presence of FITC‐PCs. Gating strategy was established as follows: a cell gate was first applied using the FSC and SSC to gate out free PCs and debris, and the macrophage only and PC only controls were then used to create a loaded macrophage gate based on FITC signal whilst excluding aggregated PCs found within the cell gate. Data were collected using a MACSQuant Flow Cytometer and analyzed using FlowJo (Version 10.7).
7
Comprehensive Immune Cell Phenotyping
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Splenocytes were stained with LIVE/DEAD fixable viability dye (Thermo Fisher Scientific), Fc blocked (clone 2.4G2, produced in house), and stained with antibodies to CD4, CD8α, CD19, CD44, CD45.1, CD45.2, CD62L, CD90.2, NK1.1, PD-1, 2B4, TCRβ, IFNγ, IL-2, Ki-67, T-bet, Eomes, and/or TNFα (BD Biosciences, eBioscience, BioLegend, and Miltenyi Biotec). H-2DbGP33−41 MHC-peptide complexes were provided as fluorophore-conjugated tetramers by E. John Wherry. Cells were fixed and permeabilized using Cytofix/Cytoperm (BD Bioscience) or the Foxp3/Transcription Factor Fixation/Permeabilization kit (eBioscience. Data were acquired using a Miltenyi Biotec MACSQuant flow cytometer and analyzed using FlowJo software versions 9.8, 9.9, or X.07 (FlowJo, LLC).
8
Calreticulin Surface Expression Analysis
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Following treatments, cells were transferred to a 96-well plate and washed twice with 150 µl/well of cold (4 °C) PBS. Then, they were resuspended in 50 µl/well of anti-CALR antibody (Abcam, Cambridge, UK) diluted in PBS supplemented with 1% bovine serum albumin (BSA) and incubated for 45 min on ice. Afterwards, cells were washed twice with 150 µl/well of cold PBS and resuspended in 50 µl/well of goat anti-rabbit IgG-Alexa Fluor 488 or -Alexa Fluor 647 (Invitrogen) diluted in PBS/1% BSA. After 30 min of incubation on ice and two additional washing steps, cells were resuspended in 120 µl/well of cold PBS supplemented with DAPI at 1 µg/mL [61 (link), 62 (link)]. Cells were analyzed by means of a MACSQuant flow cytometer and data were analyzed with FlowJo software.
9
Investigating Tick-Host Immune Interactions
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C57BL/6J mice were injected intraperitoneally (i.p.) with 500 µg of InVivoMAb anti-mouse TCR γ/δ clone UC7-13D5 (BioCell BE0070) or InVivoMAb polyclonal Armenian hamster IgG (BioCell BE0091; control) on the day (day -1) and on the morning (day 0) prior to tick placement. Ticks were microinjected with vamp33 si or vamp33 sc. Ticks fed on γδ T cell-depleted and isotype control mice for 3 days. Mice were euthanized on the third day. Tick weights were measured. Depletion was confirmed through FACS measurement of γδ T cells in the skin and spleens. Skin were labeled using VioBlue anti-CD45 (Miltenyi Biotec 130-119-130; 3 ng/μl), FITC anti-TCRγ/δ (Miltenyi Biotec 130-104-015; 3 ng/μl), PE/Vio770 CD3 (Miltenyi Biotec 130-116-530; 3 ng/μl), and PE anti-TCR β Chain (BD 553172; 2 ng/μl). Live and dead cells were discriminated using propidium iodide solution (Miltenyi Biotec). T cell populations were measured with a MACSQuant flow cytometer and analysis was performed using FlowJo software v 10.6.1.
10
Isolation of Skin Cells from Ear Samples
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Ear skin samples were digested in 1 mL of Dulbecco modified Eagle medium containing 20 mmol/L HEPES, 0.025 mg/mL Liberase, 396 U/mL DNase I, and 0.5 mg/mL hyaluronidase at 378C at 1400 rpm for 1 hour to isolate skin cells. Samples were passed through a 40-mm sieve, and the cell suspension was washed twice with PBS. For flow cytometry, skin or LN cells were resuspended in PBS/2% BSA. Cells (2-5 3 10 5 ) were stained with a mAb in 100 mL of staining solution for 30 minutes at 48C. Cell suspensions were washed twice and resuspended in 200 mL of PBS/2% BSA. For fluorescence-activated cell sorting analysis of intracellular cytokines, cells were incubated with Brefeldin A in RPMI 1640 with 10% FCS and 1% penicillin/streptomycin before fixation in forkhead box protein 3 Staining Buffer (eBioscience, San Diego, Calif) and staining. Analysis was done with the Miltenyi MacsQuant flow cytometer with MacsQuant or FlowJo analysis software (FlowJo, Ashland, Ore).
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