Fluorochrome conjugated antibodies were purchased from BD Biosciences, Biolegend, R&D Systems, and ThermoFisher Scientific. Staining with biotinylated recombinant Ly49A or Ly49C was performed as described previously (8 (link)). LCMV tetramer staining was performed as previously described (9 (link)). Melanoma-specific cells were detected with H2-Db GP100 and H2-Db Trp1 tetramers (NIH Tetramer Core Facility). All flow cytometric experiments (data acquisition) were performed using BD Fortessa II flow cytometer and FACS-Diva software (BD Biosciences) and analyzed by Flowjo v.10.3 software (TreeStar).
Fortessa 2 flow cytometer
Manufactured by BD
Sourced in United Kingdom
The BD Fortessa II is a flow cytometer designed for multiparameter analysis of cells and particles. It is equipped with multiple lasers and detectors to enable simultaneous measurement of various cellular properties, such as size, granularity, and fluorescent labeling.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (1 mentions)
Fluorochrome conjugated antibody, by BD (1 mentions)
Xvivo 15 media, by Thermo Fisher Scientific (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide escherichia coli 0111 b4, by Merck Group (1 mentions)
Zombie aqua, by BioLegend (1 mentions)
Mouse serum, by Merck Group (1 mentions)
Live dead yellow, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Worthington (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Ack buffer, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Promega (1 mentions)
Zombie yellow fixable viability kit, by BioLegend (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Lullaby reagent, by Ozyme (1 mentions)
8 protocols using fortessa 2 flow cytometer
1
Multiparameter Flow Cytometric Analysis
2
Immune Response to Heparin Therapies
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Peripheral blood mononuclear cells were prepared and cultured in serum-free X-VIVO 15 media (Life Technologies, Grand Island, New York) with UFH, branded enoxaparin (Lot number EEA1412E), and the US-generic enoxaparins produced by Sandoz (Lot number 917499) and Amphastar (Lot number EP002B2) at a dose of 10 µg/mL for 1 or 5 days. Platelet factor 4 alone (5 and 10 µg/mL) was used as the negative control. As a positive control, 50 ng/mL of the toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS; Escherichia coli 0111: B4 lipopolysaccharide, Sigma-Aldrich, St Louis, Missouri), and 10 µg/mL of the TLR7/8 agonist, R848 (Invivogen, San Diego, California), were added to the constructs. The cells were harvested, washed, and labeled for viability with LIVE/DEAD Aqua (Invitrogen, Eugene, Oregon). The cells were then labeled with a multicolor antibody panel specific for cluster of differentiation (CD) 14, human leukocyte antigen-DR, antigen-presenting cell (APC) activation/maturation markers (CD86 and CD83), and lymphocyte markers (CD3 and CD19). All antibodies were purchased from eBiosciences (San Diego, California) or BD/Biosciences (San Jose, California). Data were acquired on a BD FORTESSA II flow cytometer (BD/Biosciences) and analyzed using FlowJo software (TreeStar Inc, Ashland, Oregon).
3
Isolation and Characterization of PBMCs from Parkinson's Patients
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Blood samples for peripheral blood mononuclear cell (PBMC) isolation from matched Parkinson’s disease and control pairs were collected and processed on the same day and the same time (see Supplementary Materials and Methods for further details). PBMCs were isolated from heparinized blood using a Ficoll gradient within 2 h of phlebotomy. Isolated PBMCs were left to block in buffer for 10 min at 4°C (0.1% BSA, Probumin. Millipore, cat. no. 82-045-1; 0.01% sodium azide, Sigma-Aldrich, cat. no. S2002 made up in PBS; 2% mouse serum, Sigma-Aldrich, cat. no. M5905). Next, 0.5 μL of zombie aqua (Biolegend 423101) was added and incubated for 15 min prior to addition of surface staining antibodies (see Supplementary Methods ). Samples were incubated at 4°C for 30 min. The PBMCs were then washed twice before being fixed in 2% paraformaldehyde (PFA) for 20 min. Flow cytometry was run within 2–4 h. Flow Cytometry Standard (FCS) output files were imported into FlowJo software (version 10.5.0), which was used for gating of the cell populations, which was done blinded to group. All flow cytometry was run on the BD Fortessa II flow cytometer with a minimum of 5000 B cells recorded.
4
Co-culturing Heterotrophs with Synechocystis
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Heterotrophic bacterial isolates Acidovorax sp., Pischinibacter sp., and Staphylococcus sp. were precultured in R2A medium from single colonies overnight. Cultures were centrifuged, and the pellet was washed in fresh BG11 medium. These heterotrophs were then co-inoculated with Synechocystis in BG11 medium, where the heterotrophs were inoculated 1:10,000 from their preculture into 24-well plates with vented lids. Synechocystis was inoculated axenically in 12 additional wells. Cultures were grown for 10 days, growth was measured with OD600, and the culture medium was sampled and replenished at days 4 and 7, as described above. After 10 days, the percentage of the heterotroph population was measured using flow cytometry. Cultures were diluted 1:100 in 200 µL of BG11 with 1:5,000 SYTO40. After 20 minutes, samples were diluted 1:10 again into BG11 medium. A BD Fortessa II flow cytometer was used to count cells using the B690 filter (chlorophyl autofluorescence) and V450 filter (SYTO40). Events were plotted against these axes to separate signal from the cyanobacteria and heterotrophs.
5
Multicolor Flow Cytometry for Immune Profiling
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Briefly, cells were labeled with fluorochrome-conjugated antibodies in Phosphate-buffered saline (PBS) with Bovine Serum Albumin (BSA) (0.5%) for 10 min at 4°C. Antibodies (BD Biosciences, Oxford, UK) were as follows: CD3 (SK7), CD56 (B159), CD107a (HA4A3), NKG2D (BAT221, Miltenyi Biotec, Bisley, UK). Viability was assessed using Annexin V and 7-AAD. For quantitation of CD107a, cells were re-suspended in complete media containing 100 ng/ml PMA, 1 μg ionomycin and 0.1% 2-mercaptoethanol (stimulated) or complete media with 0.1% 2-mercaptoethanol (non-stimulated) for 2 h at 37°C. Fluorescence minus one (FMO) controls (where samples are stained sequentially with all antibodies except one) were used to set gates. Acquisition was performed using a Fortessa II flow cytometer (BD Biosciences, Oxford, UK) and analysis was carried out using FlowJo version 10.5.0 (Tree Star Inc., OR, USA). The gating strategy used for analysis of lymphocyte subtypes was performed as described in our previously study (12 (link)) and is shown in Supplementary Figure S1 .
6
Detailed Immune Cell Profiling of PBMCs
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Frozen PBMC samples were thawed and stained with Fixable viability dye (Yellow Live/Dead, Thermo Fisher Scientific) followed by two different panels of membrane markers. Two different panels were used, a simple one for the discovery cohort and a more thorough panel for the validation cohort (full list of both antibody panels in Supplementary file 4 and Supplementary file 5 ). These two panels allow definition of immune cell populations as described in Figure 1—figure supplement 2 . Patients’ samples and corresponding Fluorescence Minus One (FMO) Controls were acquired in a Fortessa II flow cytometer (BD, Berkshire, UK) and analysed with FlowJo software (Tree Star).
7
Dissociation and Flow Cytometry of Adult Testes
8
Homologous Recombination and NHEJ Assays
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Reporter cell lines were used to assess homologous recombination (DR-GFP) and non-homologous end joining (EJ5-GFP) [13] (link). For transfections, I-SceI expression vector (pCBASce), GFP expression vector (pCAGGS-NZEGFP) and a control empty vector (pCAGGS-BSKX) were used [13] (link). In detail, 2 × 105 cells were plated into a 12-well dish and transfected with non-targeting or siRNA targeting ATMIN (Dharmacon) using Lullaby reagent (OZ Biosciences). After 48 h, cells were transfected with I-SceI or control plasmids. Transfection complexes were prepared by mixing Lipofectamine 2000 (Life Technologies) in OptiMEM (Invitrogen) with 0.8 μg of expression vectors for I-SceI or control vectors per sample. Samples were analyzed 3 days after transfection by immunoblotting to assess the knockdown efficiency. The frequency of GFP+ cells was determined on a Fortessa II flow cytometer (BD Bioscience).
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