Cells were co-transfected with pNFAT-TA-mRFP (for NFAT-driven RFP expression) and pOTTC589- pAAV c-fos Nuc-eYFP using the Amaxa system. Twenty-four hours after transfection, cells were treated with different concentrations of thapsigargin (as stated in the text) for 40 min and then washed several times in medium without stimulus. After 12 h incubation, cells were fixed with 4% paraformaldehyde and washed three times with PBS. The YFP and RFP expression in individual cells were detected using a BD X-20 flow cytometer. Data were analysed using FlowJo software.
X 20 flow cytometer
Manufactured by BD
The BD X-20 flow cytometer is a laboratory instrument designed to analyze and sort cells or particles in a fluid sample. It utilizes the principles of flow cytometry to provide quantitative and qualitative data about the physical and biochemical characteristics of individual cells within a heterogeneous population.
Automatically generated - may contain errors
Lab products found in correlation
Zombie nir fixable viability kit, by BioLegend (2 mentions)
Streptavidin pe, by BioLegend (2 mentions)
Clones fn50, by BioLegend (2 mentions)
Zombie uv fixable viability kit, by BioLegend (2 mentions)
Lenticrispr v2, by Addgene (1 mentions)
Ebioscience fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Ls magnetic column, by Miltenyi Biotec (1 mentions)
Efluor 780, by Thermo Fisher Scientific (1 mentions)
Brilliant violet buffer, by BD (1 mentions)
Saponin, by Merck Group (1 mentions)
Live dead dye, by Thermo Fisher Scientific (1 mentions)
Foxp3 buffer set, by BD (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
Fortessa, by BD (1 mentions)
Phosflow lyse fix buffer, by BD (1 mentions)
Phosflow perm wash buffer, by BD (1 mentions)
Protein labeling kit, by Thermo Fisher Scientific (1 mentions)
10 protocols using x 20 flow cytometer
1
NFAT-Driven RFP Expression in Cells
2
CRISPR-Mediated Knockout of Glycosylation Genes in BV2 Cells
3
Multiparametric Flow Cytometry Analysis
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Spleens harvested from infected animals were meshed, and RBCs were removed using ammonium-chloride-potassium (ACK) buffer (Lonza). Cells were stained with fluorochrome-conjugated antibodies and analyzed in a BD X20 flow cytometer. Flow cytometry data were analyzed in FlowJo software v10. For histological analyses, whole brains and spleens were harvested, fixed in 10% buffered formalin, and embedded in paraffin. Brain sections were stained with hematoxylin and eosin (H&E). Spleen sections were stained with PNA and IgM and visualized using the strategy outlined in reference 48 (link).
4
Tetramers for Cell Staining and Analysis
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Tetramers were produced in-house using refolded monomeric, biotinylated pMHC, and streptavidin-PE (Biolegend) at a 1:4 molar ratio. streptavidin-PE was added in 10 steps and incubated for 10 min while shaking at room temperature. Insoluble proteins were removed by brief centrifugation at 13,000 g and 0.05–0.1% sodium azide added for preservation. Tetramers were kept for up to 3 months at 4°C. Cells were stained for CD69 with clones FN50 (Biolegend). Staining for CD45 (clone HI30; Biolegend) was used to distinguish target and effector cells in co-culture assays with U87 cells. Cell viability staining was routinely performed for plate stimulations and U87 co-culture using fixable violet or near-infrared viability dyes (Zombie UV fixable viability kit [Biolegend], Zombie NIR fixable viability kit [Biolegend], eBioscience fixable viability dye eFluor 780 [Invitrogen]). Samples were analysed using a BD X-20 flow cytometer, and data analysis was performed using FlowJo v10 (BD Biosciences).
5
Tetramer-based Enrichment and Analysis
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Splenocytes was harvested from naive C57BL/6 or MD4 mice. Cells were then incubated for 30 minutes with a mixture of tetramers consisting full length recombinant CSP conjugated to PE and domain specific region of CSP, conjugated to APC. The cells were washed to remove unbound tetramers, and were enriched for PE after incubating with anti-PE microbeads and passing over the LS magnetic column (Miltenyi) according to the manufactures recommendation. Subsequently, antibody staining was performed on the enriched cells as per established protocol. Prior to acquisition on a BD X-20 flow cytometer, 1 × 104 CountBright absolute counting beads (Invitrogen) were added, to normalize the number of tetramer specific cell events obtained in each sample.
6
Tetramer Staining for Flow Cytometry
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Tetramers were produced in-house using refolded monomeric, biotinylated pMHC and streptavidin-PE (Biolegend) at a 1:4 molar ratio. streptavidin-PE was added in 10 steps and incubated for 10 min while shaking at room temperature. Insoluble proteins were removed by brief centrifugation at 13,000 g and 0.05-0.1 % sodium azide added for preservation. Tetramers were kept for up to 3 months at 4°C. Cells were stained for CD69 with clones FN50 (Biolegend). Staining for CD45 (clone HI30; Biolegend) was used to distinguish target and effector cells in co-culture assays with U87 cells. Cell viability staining was routinely performed for plate stimulations and U87 co-culture using fixable violet or near-infrared viability dyes (Zombie UV fixable viability kit [Biolegend], Zombie NIR fixable viability kit [Biolegend], eBioscience fixable viability dye eFluor 780
[Invitrogen]). Samples were analysed using a BD X-20 flow cytometer and data analysis was performed using FlowJo v10 (BD Biosciences).
[Invitrogen]). Samples were analysed using a BD X-20 flow cytometer and data analysis was performed using FlowJo v10 (BD Biosciences).
7
Probing Antigen-specific B Cell Responses
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The antigen-specific B cell responses were probed by fluorochromes labeled DENV1 and DENV2 E proteins as previously described (24 (link)). Cell were analyzed on an X20 flow cytometer (BD Biosciences) and data processed using FlowJo version 10.6.0 (Tree Star).
8
Multiparametric Flow Cytometry Analysis
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Multiparametric flow cytometry was used for phenotypic and functional analysis of PBMCs and IHLs. Cells were stained with a fixable Live/Dead dye (Invitrogen) before incubation with saturating concentrations of surface mAbs diluted in 50% Brilliant violet buffer (BD) and 50% PBS for 30 min at 4°C. See also Table S1 for full details regarding antibodies used. Cells were fixed and permeabilized for further functional assessment with either Cytofix/Cytoperm (BD) or FoxP3 Buffer Set (BD) according to the manufacturer’s instructions. Saturated concentrations of mAbs for 30 min at 4°C were diluted in 0.1% saponin (Sigma-Aldrich) for the detection of intracellular proteins or in 1× PBS for the detection of intranuclear proteins. All samples were acquired on either an LSRII or X20 flow cytometer (BD) and analyzed using FlowJo (Tree Star).
9
Isolation and Characterization of Murine B Cells
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Spleens were collected from mice after cervical dislocation, and single cell suspension was prepared after passing through a 70 μM cell strainer into FACS buffer. Cells were briefly blocked for 30 minutes using 1μg/mL Streptavidin and 10 μg/mL TruStain fcX antibody diluted in FACS buffer. An antibody master mix was prepared in FACS buffer to stain the surface antigens of cells as per standard protocol using antibodies and B cell tetramers (prepared in house) for 30 minutes in dark. Cells were then lyzed using ACK lysis buffer and washed twice before staining with 1% 7AAD as Live/dead dye. Samples were acquired with a BD Fortessa or X20 flow cytometer, and data was analyzed using FlowJo software. The universal gating strategy is shown Figure S2 , after which the further gating to study the B cells of interest is provided in the results.
For adoptive transfer experiments with Ighg2A10 and B1-8 cells, single cell suspensions of splenocytes were prepared and an aliquot used for flow cytometry using probes as described below to determine the proportion of cells that were antigen-specific B cells. The concentration of splenocytes was then normalized to allow for the transfer of the desired number of antigen specific cells.
For adoptive transfer experiments with Ighg2A10 and B1-8 cells, single cell suspensions of splenocytes were prepared and an aliquot used for flow cytometry using probes as described below to determine the proportion of cells that were antigen-specific B cells. The concentration of splenocytes was then normalized to allow for the transfer of the desired number of antigen specific cells.
10
Measurement of Antigen-Specific B-Cell Responses
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To measure the antigen-specific B-cell responses, we used protein labeling kits (Thermo Fisher Scientific) to conjugate Alexa Flour 647 (AF647) and Alexa Flour 548 (AF548) to extracellular domain of DENV1 (DENV1/VN/BID-V949/2007) and DENV2 (DENV2/GWL39 IND-01) E proteins (~50 kDa, CTK Biotech), respectively. One million splenocytes were incubated with DENV1/E-AF647 and DENV2/E-AF548 probes on ice for 30 min in the dark. The cells were then surface stained with conjugated anti-CD90.2, anti-F4/80, anti-CD11c, anti-CD4, anti-CD8, anti-Ly6G, anti-NK1.1, anti-IgM, anti-IgD, anti-GL7, anti-CD45R, anti-CD38 antibodies (Supplementary Table 1 ), and LIVE/DEAD stain FSV780 (BD Biosciences) on ice for 30 min. For the intracellular staining, cells were fixed and permeabilized with Phosflow Lyse/Fix buffer (BD Biosciences) for 10 min at 37 °C in the dark and subsequently incubated with Phosflow Perm/Wash buffer for 30 min at room temperature. Then, cells were stained with anti-Ig (H+L) and anti-Bcl6 antibodies for 30 min at 4 °C in the dark. Cells were analyzed on an X20 flow cytometer (BD Biosciences) and data processed using FlowJo version 10.6.0 (Tree Star).
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