Cytokine assays were performed using the Milliplex® Map Mouse Cytokine/Chemokine Magnetic Bead Panel kit (Millipore, Billerica, MA, USA) and Milliplex™ Map TGFβ1 Single Plex Kit on a Luminex® 200 multiplex system (Invitrogen, Carlsbad, CA, USA). According to the manufacturer’s instructions, we prepared all reagent dilutions (beads, cytokine standards, cytokine controls, biotinylated detection antibody, etc.). According to the vendor-provided instructions, we performed cytokine assays. We incubated the cytokine standards, controls, samples, and microspheres on a rotator in 96-well filter bottom microtiter plates (Millipore) at RT for 2 h to allow for subsequent washing. After an additional 30 min incubation on the rotator and washing, we added 100 µL of streptavidin (10 µg/mL)-conjugated R-phycoerythrin (Invitrogen) to each well. After a 30 min incubation and a final wash, we resuspended the microspheres in 200 µL of washing buffer, and placed the 96-well microplate in a Luminex 100 instrument. We determined the amount of cytokine bound to the microsphere as the median fluorescence intensity (MFI) of the reporter molecule, phycoerythrin. Then, the MFI of unknown samples was converted into picograms per milliliter based on the known cytokine concentrations of the standard curve using a five-parameter regression formula.
R phycoerythrin
Manufactured by Thermo Fisher Scientific
Sourced in United States
R-Phycoerythrin is a natural fluorescent protein derived from red algae. It is a photosynthetic pigment that functions as a light-harvesting chromoprotein in photosynthetic organisms. R-Phycoerythrin exhibits bright red fluorescence and can be used as a fluorescent label or reporter in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Keyhole limpet hemocyanin (klh), by Merck Group (2 mentions)
Chicken ovalbumin, by Merck Group (2 mentions)
Hen egg lysozyme, by Merck Group (2 mentions)
Human serum albumin (hsa), by Merck Group (2 mentions)
Ultra pure rabbit serum albumin, by Merck Group (2 mentions)
Pd 10 size exclusion column, by GE Healthcare (2 mentions)
Asmtpa gf ht, by DiaSorin (2 mentions)
Digitonin, by Thermo Fisher Scientific (2 mentions)
Digitonin, by Merck Group (2 mentions)
360kd polyvinylpyrrolidone pvp, by Merck Group (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
Milliplex map mouse cytokine chemokine magnetic bead panel kit, by Merck Group (1 mentions)
Facsaria 3 sorter, by BD (1 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Viability stain, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Fluoromount g mounting medium with dapi, by Thermo Fisher Scientific (1 mentions)
Axiovision software, by Zeiss (1 mentions)
16 protocols using r phycoerythrin
1
Multiplex Cytokine Quantification
2
Isolation of CD44+/CD24low/- HeLa cells
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The HeLa-SFCs were dispersed into single cell suspensions and stained with antibodies specific for CD44 conjugated with fluorescein isothiocyanate (FITC, Invitrogen) and for CD24 conjugated with Rphycoerythrin (Invitrogen). Immunoreactive HeLa-SFCs were subject to fluorescence-activated cell sorting analysis using an FACSAria III sorter (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturers' instructions, after which the cells exhibiting an expression pattern of CD44+/CD24low/− were identified accordingly (15 (link)).
3
Detecting SARS-CoV-2-specific Memory B Cells
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Fluorescent SARS-CoV-2-specific S6P (87 (link)) (provided by Roland Strong, Fred Hutchinson Cancer Research Center, Seattle, WA) and RBD probes were made by combining biotinylated protein with fluorescently labeled streptavidin. The S6P probes were made at a ratio of 1:1 molar ratio of trimer to SA. Two S6P probes, one labeled with AlexaFluor488 (Invitrogen), one labeled with AlexaFluor647 (Invitrogen), were used in this panel in order to increase specificity of the detection of SARS-CoV-2-specific B cells. The RBD probe was prepared at a 4:1 molar ratio of RBD monomers to SA, labeled with R-phycoerythrin (Invitrogen). Cryopreserved PBMCs from SARS-CoV-2-convalescent participants and a pre-pandemic SARSCoV-2-naïve donor were thawed at 37°C and stained for SARS-CoV-2-specific memory B cells with a flow cytometry panel shown in Supplementary Table 3 . Cells were stained first with the viability stain (Invitrogen) in PBS for 15 min at 4°C. Cells were then washed with 10% FBS/PBS and stained with a cocktail of the three probes for 30 min at 4°C. The probe cocktail was washed off with 10% FBS/PBS and the samples were stained with the remaining antibody panel and incubated for 25 min at 4°C. The cells were washed two times and resuspended in 1% paraformaldehyde/1x PBS for collection on an LSR II flow cytometer (BD Biosciences). Data was analyzed in Flow Jo version 9.9.4.
4
Multiplex Immunofluorescence Microscopy
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To enable double labeling, immunofluorescence was performed following the same steps as conventional immunohistochemistry regarding the application of the primary antibodies. Subsequently, sections were stained with specific fluorophore-conjugated secondary antibodies, Alexa Fluor 488 (Invitrogen), R-phycoerythrin (PE; Invitrogen), and DyLightTM 633 (ImmunoReagents-Raleigh, NC, USA). Fluoromount-G Mounting Medium with DAPI (Invitrogen) was used to mount the slide and coverslip. Images were captured on a Zeiss Axio lmager M1 microscope with the AxioVision software (Carl Zeiss Microscopy, LLC, White Plains, NY, USA).
5
Quantitative Bacterial Surface Labeling
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An aliquot of approximately 108 recombinant bacteria was added to 1×PBSP (phosphate-buffered saline (PBS) with 0.1% Pluronic F108 NF surfactant (BASF Corporation, Mount Olive, NJ)), pelleted by centrifugation (15000 g, 6 min, 4°C), re-suspended in 22 nM biotinylated human polyclonal IgG, and incubated at room temperature with gentle mixing for 45 min. Cells were then washed with ice-cold 1xPBSP, re-suspended in 10 μg/ml streptavidin conjugated with R-Phycoerythrin (Invitrogen) and 150 nM Alexa Fluor 647-Human Serum Albumin (HSA) conjugate, and incubated on ice for 30 min. The samples were then washed with ice-cold 1×PBSP, resuspended in 1×PBSP, and analyzed using a Gallios™ flow cytometer (Beckman Coulter, Inc., Indianapolis, IN, USA).
6
Custom Coronavirus Multiplex Array
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A custom coronavirus multiplex array (71 (link)) was performed using SARS-S1 subunit (S1N-S52H5, Acrobiosystems), SARS-CoV-2 S1 (40591-V08B1) and HCoV NL63 S1 and S2 subunits (40604-V08B, Sino Biological), NL63 S trimer [100788, bpsbioscience], and hexahistidine tagged RBD WT (SARS CoV-2, isolate Wuhan-Hu-1, NCBI Reference Sequence: YP_009724390.1, aa residues 319-541 (72 (link)),) and 24 variants identified from the GISAID RBD surveillance repository (71 (link)). TrACE2-Fc was biotinylated using EZ-Link® Sulfo-NHS-LC-Biotin (ThermoFisher Scientific) according to the manufacturer’s instructions. Biotinylated trACE2-Fc (70 nM) was incubated with a concentration series, eight two-fold dilutions from 282 nM, of unlabelled trACE2-Fc, flACE2-Fc, EflACE2-Fc fusion proteins or the inhibitory human mAb S35 (AcroBiosystems) and binding to RBD or S proteins coupled to beads was determined using first Streptavidin, R-Phycoerythrin Conjugate (SAPE) (Thermo Fisher) at 4µg/ml (1 h), followed by 10µg/ml of R-Phycoerythrin, Biotin-XX Conjugate (Thermo Fisher) (1 h) and multiplex analysis. Apparent IC50 (nM) values are indicated from curve fits.
7
Fluorescent Protein Conjugation Protocol
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Ultra-pure rabbit serum albumin, human serum albumin, chicken
ovalbumin, Hen egg lysozyme, and keyhole limpet hemocyanin were purchased
from Sigma (seeKEY RESOURCES TABLE ).
R-Phycoerythrin was purchased from ThermoFisher (seeKEY RESOURCES TABLE ). All carrier proteins were
resuspended in sterile PBS according manufacturers instructions.
Selected carriers were conjugated to fluorescein isothiocyanate
obtained from Sigma (seeKEY RESOURCES
TABLE ) through simple amine modification. Briefly, carrier
proteins were dissolved in 0.1M NaHCO3 and mixed with a
pre-determined amount of FITC dissolved in DMSO. Reaction tubes were rotated
for 2h at room temp. Total reaction volumes were loaded onto PD-10 size
exclusion columns obtained from GE healthcare, and unlabeled FITC was
removed as per manufacturers instructions. Labeled protein was assessed for
conjugation ratio through spectral analysis, and concentrated to desired
stock concentration.
ovalbumin, Hen egg lysozyme, and keyhole limpet hemocyanin were purchased
from Sigma (see
R-Phycoerythrin was purchased from ThermoFisher (see
resuspended in sterile PBS according manufacturers instructions.
Selected carriers were conjugated to fluorescein isothiocyanate
obtained from Sigma (see
TABLE
proteins were dissolved in 0.1M NaHCO3 and mixed with a
pre-determined amount of FITC dissolved in DMSO. Reaction tubes were rotated
for 2h at room temp. Total reaction volumes were loaded onto PD-10 size
exclusion columns obtained from GE healthcare, and unlabeled FITC was
removed as per manufacturers instructions. Labeled protein was assessed for
conjugation ratio through spectral analysis, and concentrated to desired
stock concentration.
8
Quantifying Murine tPA Protein in Brain
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To measure total murine tPA protein from tPABAC-Cer brain extracts, 50 μg of Rabbit anti-murine tPA (mtPA; Molecular Innovations, ASMTPA-GF-HT) was coupled to Luminex carboxylated beads for mtPA capture. Standards of known concentration of murine tPA (Molecular Innovations, MTPA) and brain extract samples [diluted in 0.4 M HEPES, 0.1 M NaCl (pH7.4), and 1.0% Triton X-100] were loaded onto a 96 well filter plate (Millipore) and incubated with 5000 beads [PBS-1.0% bovine serum albumin (BSA)] for 2 h at room temperature in the dark. The solution from was removed from each well and washed twice with PBS-0.05% Tween 20. The beads were then mixed with continuous shaking in the dark at room temperature for 1 h with 2 μg/ml biotin-labeled rabbit anti-mouse tPA-high titer (Molecular Innovations, ASHTPA-HT), after which 10 μg/ml of Streptavidin, R-Phycoerythrin (ThermoFisher Scientific, S866) was added to each well for 1 h. The solution was removed from each well, and the beads were washed three times with PBS-0.05% Tween 20, and, lastly, sheath fluid was added for 5–10 min. The beads were then read with the Luminex 100 (medium setting; 10-μl sample size; 100 events/bead).
9
Membrane Permeabilization and Nuclear Integrity
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Cells were permeabilized as previously described (Adam et al., 1992 (link)). Cells were incubated on ice for 5 min. Then cells were washed in cold transport buffer (TB) (20 mM HEPES, 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, and 1 mM ethylene glycol tetraacetic acid (EGTA) at pH 7.3). Cells were then incubated on ice in TB with 34 µg/mL digitonin (Sigma) for 5 min. Cells were then washed twice in cold TB and twice in 37C TB with 1.5% (wt/vol) 360kD polyvinylpyrrolidone (PVP, Sigma). Cells were imaged in 37C TB with 1.5% PVP. In order to confirm that 34 µg/mL digitonin left the nuclear envelope intact but resulted in the permeabilization of the plasma membrane, cells were incubated with 500 ng/mL R-phycoerythrin (ThermoFisher, Waltham, MA) for 10 min and then imaged. R-phycoerythrin is a 240 kD, so it should not readily diffuse through the NPC and can be used as a marker for nuclear integrity.
10
Fluorescent Protein Conjugation Protocol
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