From splenocytes, T cells and B cells were isolated using a CD4 T cell or CD19 B cell MACS isolation kit, respectively, with an AutoMACS separator (Miltenyi Biotech) according to the manufacturer’s instructions. CD226+CD4+ T cells were further purified using a FACSAria IIu cell sorter (BD Biosciences). For CNS cells, CD45hiCD19+ MHC II+ B cells, CD45hiCD19−MHC II+CD11c+CD317hiB220+ pDCs, CD45hi TCR-β+CD4+ T cells, and CD45intCD11b+ microglia cells were sorted using a FACSAria IIu cell sorter (BD Biosciences).
Facsaria iiu cell sorter
Manufactured by BD
Sourced in United States
The FACSAria IIu cell sorter is a high-performance flow cytometry instrument designed for cell sorting applications. It utilizes advanced optical and fluidic systems to enable efficient and precise separation of cells based on their physical and fluorescent properties.
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Lab products found in correlation
Facsaria iiu, by BD (4 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
Dnase 1, by Merck Group (3 mentions)
Lsr iiu, by BD (3 mentions)
Facscalibur analyzer, by BD (2 mentions)
Dispase, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Roche (2 mentions)
Lsr 2 flow cytometer, by BD (2 mentions)
Neural tissue dissociation kit, by Miltenyi Biotec (2 mentions)
Myelin debris removal solution, by Miltenyi Biotec (2 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (2 mentions)
Fc block, by Thermo Fisher Scientific (2 mentions)
Cytofix, by BD (2 mentions)
Cytoslides, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lymphoprep, by Axis-Shield (2 mentions)
Facscalibur flow cytometer, by Tree Star (2 mentions)
Percp conjugated antihuman cd4 clone sk3, by BD (2 mentions)
Macs column, by Miltenyi Biotec (2 mentions)
Ionomycin, by Merck Group (2 mentions)
66 protocols using facsaria iiu cell sorter
1
Isolation of Immune Cell Subsets
2
Ramp1 Knockout T Cell Antitumor Response
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Total CD8+ T cells were isolated from the spleen of wild-type (CD45.1+) or Ramp1−/− (CD45.2+) mice, expanded and stimulated in vitro using a mouse T cell Activation/Expansion Kit (Miltenyi Biotec. #130-093-627). CD8+ cells from Ramp1−/− and Ramp1WT were injected separately or 1:1 mix through tail vein of Rag1−/− mice. One week after, the mice were inoculated with B16F10-mCherry-OVA cancer cells (5 × 105 cells; i.d.), and tumour growth was measured daily using a handheld digital caliper. On day 10, tumours were collected and Ramp1−/− (CD45.2+) and Ramp1WT (CD45.1+) CD8+ T cells were immunophenotyped using a FACSCanto II (Becton Dickinson) or FACS purified using a FACSAria IIu cell sorter (Becton Dickinson).
3
Adoptive Transfer of Naive CD8+ T Cells
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CD8+ T cells were isolated from OT-I mice spleens and magnet sorted (StemCell; 19858). Naive CD8+ T cells (CD8+CD44lowCD62Lhi) cells were then purified by FACS using an FACSAria IIu cell sorter (Becton Dickinson) and injected (1 × 106 cells, i.v., tail vein) into vehicle- or RTX-exposed Rag1−/− mice.
4
Isolation and Purification of Endothelial Cells from Kidneys
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For purification of EC from kidneys, we combined MACS-based pre-enrichment and FACS strategies. Single-cell suspensions were prepared from kidneys as described above. After Fc block and subsequent staining with anti-CD31-PE and anti-CD45-FITC, cells were washed and incubated with 1:5 diluted anti-PE microbeads (Miltenyi Biotech, 130-048-801) for 15 min. Positive selection of magnetically labeled cells was performed using LS columns and MidiMax Separator in combination with MACS Multistand (all from Miltenyi Biotec) according to manufacturer’s instructions. After elution from columns, cells were pelleted, filtered through 40 µm mesh and proceeded for sorting on a FACSAria IIu cell sorter (Becton-Dickinson). Total yield of EC (CD31+CD45− cells) was about 5 × 105 to 1 × 106 live cells per kidney. RNA was isolated directly after sorting using a Qiagen RNEasy micro kit according to the manufacturer’s protocol.
5
Annexin V Apoptosis Assay in A549 Cells
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A549 cells were infected with PR8 at an MOI of 2. Cells were stained with Annexin V Apoptosis Detection Kit FITC according to manufacturer’s procedure (eBioscience) at the indicated time points post-infection. Apoptotic and non-apoptotic cells were sorted with a FACSAria IIu cell sorter (BectonDickinson).
6
Lentiviral Transduction and Cell Sorting
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Lentiviral transduced RAW 264.7 and dendritic D1 cells were sorted by eGFP fluorescence with a FACSAria IIu cell sorter (Becton Dickenson). RAW 264.7 cells were sorted three times and D1 cells twice. Non-transduced RAW 264.7 or D1 cells were applied as negative control. After sorting, cells were cultured in medium containing 10 μg/ml gentamicin. Gentamicin was omitted during following cell passages.
Sorted cells were analysed using an LSR II (Becton Dickenson) or a BD FACSCalibure (Becton Dickenson) flow cytometer (S2 Fig ). In preparation for flow cytometry analysis, the cells were harvested, washed once with FACS buffer (PBS containing 2% FBS) and counted. Subsequently they were resuspended in 300 μl FACS buffer containing 1 μg/ml DAPI or 1 μg/ml Pacific Blue and analysed.
Sorted cells were analysed using an LSR II (Becton Dickenson) or a BD FACSCalibure (Becton Dickenson) flow cytometer (
7
Isolation and Characterization of Mouse Keratinocytes
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Keratinocytes were isolated from mouse back skin as described before (Jensen et al, 2010 (link)), washed in PBS containing 2% FCS, and incubated for 1 h at 4°C in primary antibody (see Table 1 ) in PBS 2% FCS. In case of non-fluorophore–conjugated antibodies, the cells were subsequently incubated with donkey antigoat Alexa Fluor 647 (1:200 dilution; Invitrogen) antibody for 30 min at 4°C. The cells were analyzed on a Becton Dickinson FACSCalibur analyzer after the addition of indicated life/dead cell marker. For fluorescent-activated cell sorting, GFP-positive cell population was obtained using a Becton Dickinson FACSAria IIu cell sorter.
8
Generation of Stable Cell Lines for SSTR Study
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HEK293 cells, stably expressing GloSensor-22F cAMP probe (HEK-Gs), had been generated in-house and characterized earlier 36 (link). In order to obtain double-stable cell lines, overexpressing GloSensor-22F cAMP probe and a desired SSTR subtype, HEK-Gs cells were transfected with SSTR2_HA-P2A-mCherry, SSTR3_Myc-P2A-mCherry or SSTR5_Flag-P2A-mCherry plasmids. Transfections were carried out with Xfect polymer (Clontech Laboratories, Cat#631317), according to the manufacturer`s instructions. After 4-6 weeks of continuous selection with 500 μg/ml geneticin (G418; Roche, Cat#04727878001), the evolved stable clones were further sorted at least twice with FACSAria IIu cell sorter (Beckton Dickinson; provided by Cell Imaging Core of Turku Centre for Biotechnology) to collect the brightest fraction of mCherry-positive cells. As mCherry and SSTRs are transcriptionally coupled via P2A linker 46 , both proteins are expected to accumulate in proportional amounts in the cells with the plasmid expression, which makes a rationale for the above FC-aided enrichment approach. The expression of target SSTR subtypes in the procured cultures was eventually validated with indirect immunolabelling in flow cytometry analysis, as described below. BON1 cells, stably expressing GloSensor-22F cAMP probe (BON-Gs), were derived and characterized in a similar fashion.
9
RNA-Seq Analysis of DRG Neurons and B16F10 Cells
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A total of 1 × 104 naive Trpv1cre::-CheRiff-eGFPfl/WT DRG neurons were co-cultured with 5 × 104 B16F10-mCherry-OVA cells overnight in complete DMEM (Corning, 10-013-CV) supplemented with 10% FBS (Seradigm, 3100), 1% penicillin–streptomycin (Corning, MT-3001-Cl), 0.05 ng μl−1 NGF (Life Technologies, 13257-019), 0.002 ng μl−1 GDNF (PeproTech, 450-51-10). After 48 h, the cells were detached and TRPV1 neurons (eGFP+mCherry−) and B16F10-mCherry-OVA (eGFP−mCherry+) were FACS purified using a FACSAria IIu cell sorter (Becton Dickinson), and the cell supernatant was collected for ELISAs.
RNA-sequencing libraries were constructed using the Illumina TruSeq Stranded RNA LT Kit (Illumina) following the manufacturer’s instructions. Illumina sequencing was performed at Fulgent Genetics. Reads were aligned to the mouse mm10 reference genome (GenBank assembly accession GCA_000001635.2) using STAR v.2.7 (ref. 58 (link)). Aligned reads were assigned to genic regions using the featureCounts function from subread v.1.6.4 (ref. 65 (link)). Gene expression levels were represented by TPM. Hierarchical clustering was computed using the heatmap.2 function (ward.D2 method) from the gplots R package (v.3.1.3). Differential gene expression analysis was performed using DeSeq2 v.1.28.159 (link). These data have been deposited in the NCBI’s GEO (ref. 60 (link)) (GSE205865).
RNA-sequencing libraries were constructed using the Illumina TruSeq Stranded RNA LT Kit (Illumina) following the manufacturer’s instructions. Illumina sequencing was performed at Fulgent Genetics. Reads were aligned to the mouse mm10 reference genome (GenBank assembly accession GCA_000001635.2) using STAR v.2.7 (ref. 58 (link)). Aligned reads were assigned to genic regions using the featureCounts function from subread v.1.6.4 (ref. 65 (link)). Gene expression levels were represented by TPM. Hierarchical clustering was computed using the heatmap.2 function (ward.D2 method) from the gplots R package (v.3.1.3). Differential gene expression analysis was performed using DeSeq2 v.1.28.159 (link). These data have been deposited in the NCBI’s GEO (ref. 60 (link)) (GSE205865).
10
Immunophenotyping of Cell Populations
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Cells were trypsinized, washed in PBS containing 2% FCS, and incubated for 1 h at 4 °C in primary antibody in PBS 2% FCS. Next, the cells were washed twice in PBS containing 2% FCS and incubated with PE-conjugated donkey anti-mouse (Biolegend #406421; 1:200 dilution) or donkey anti-rat (Biolegend # 406421; 1:200 dilution) antibody for 30 min at 4 °C. After subsequent washing steps, cells were analyzed on a Becton Dickinson FACS Calibur analyzer. For fluorescent-activated cell sorting, α3-negative cell population was obtained using a Becton Dickinson FACSAria IIu cell sorter.
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