All flow cytometry antibodies were purchased from BD Pharmingen (San Diego, CA, USA), BioLegend (San Diego, CA, USA) and KeyGen Biotech (Nanjin, Jiangsu, China). The following antibodies were obtained from BD Pharmingen™: CD3-APC-Cy™7, CD4-FITC, CD8-PECy™5, PD-1-APC, and LAG3-PE. The following reagents were obtained from BioLegend: PerCP/Cy5.5-conjugated anti-IL-2, PE/Cy7-conjugated anti-IL-6, PerCP/Cy5.5-conjugated anti-TNF-α, and PE/Cy7-conjugated anti-IFN-γ antibodies, a FITC Annexin V/PI kit, and a KGA: FITC-BrdU kit. The following quantum MESF beads were purchased from Bangs Laboratories: Fluorescent Microspheres, Intensity Standard: Dragon Green, Flash Red, PE-MESF, and APC-MESF. The PMA/ionomycin mixture (250X) was purchased from MultiSciences (Lianke) Biotech (Hangzhou, Zhejiang, China). Enzyme-linked immuno sorbent assay (ELISA) kits for human interleukin-2 (IL-2), IL-6, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) were purchased from Beijing 4A Biotech (Beijing, China). Brefeldin A was purchased from Qcbio Science & Technologies Co., Ltd. (Beijing, China).
Flash red
Manufactured by Bangs Laboratories
Sourced in United States
Flash Red is a compact, high-powered LED light source designed for a variety of laboratory applications. It provides a consistent, bright illumination for various imaging and analytical tasks.
Automatically generated - may contain errors
Lab products found in correlation
Dragon green, by Bangs Laboratories (2 mentions)
Pd 1 apc, by BD (1 mentions)
Lag3 pe, by BD (1 mentions)
Cd8 pe cy5, by BD (1 mentions)
Fluorescent microspheres, by Bangs Laboratories (1 mentions)
Annexin 5 fitc pi kit, by BioLegend (1 mentions)
Cd3 apc cy7, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Streptavidin coated microspheres, by Bangs Laboratories (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
Fsfr005, by Bangs Laboratories (1 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
7 protocols using flash red
1
Comprehensive Immunophenotyping Protocol
2
Streptavidin-coated Microsphere Binding Assay
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Streptavidin-coated microspheres were either colorless or Flash Red fluorescent, with mean diameters of ∼100 nm or ∼1 μm (Bangs Laboratories), respectively. Spheres were washed twice with ∼20 volumes of KMEH, resuspended in 1 volume of KMEH + 1 mg/ml bovine serum albumin, and added in 10 μl aliquots to 40 μl protein mixtures in KMEH. The protein mixture always included a saturating concentration of SpirNT-biotin (400 nM). When included, 200 nM (dimeric) CapuCT was preincubated with the SpirNT-biotin on ice for 10 min to allow the formation of SpirNT–CapuCT complexes. Protein mixtures were incubated with spheres for 10 min on ice and then spun for 10 min at 10,000× rpm and 4°C. Buffer and excess proteins (i.e., unbound SpirNT–biotin and CapuCT) were aspirated and pellets were gently resuspended in 30 μl KMEH. Pellets were briefly sonicated (∼5 s) if clumped and not well dispersed when visualized by TIRF microscopy.
3
MSCs Labeled with Paramagnetic Microspheres
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The MSCs were labeled with paramagnetic microspheres (diameter: 0.9 μm; composition: polystyrene with 62% (w/w) iron oxide; fluorescent label: Flash Red; Bangs Laboratories, Fishers, IN, USA), serving as MRI contrast agent and histological marker as described before (36 (link)). Briefly, the cells were incubated overnight with 11.2 × 106 microspheres/cm2 cell culture surface in MSC cell culture media. The MSCs were co-labeled with the fluorescent vital dye Vybrant DiI-CM (Life Technologies) following the instructions of the manufacturer to identify the transplanted MSC-containing microspheres in the histological analyses.
4
Fluorescent and Non-Fluorescent PNP Characterization
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We used fluorescent-labelled PNPs (Flash Red, excitation wavelength 660 nm, emission wavelength 690 nm) with a mean diameter of 0.513 µm (Bangs Laboratories, Inc., Fishers, IN, USA) for the microscopy observations, while non fluorescent PNPs with comparable size (diameter range=0.4-0.6 µm; Spherotech, Inc., Lake Forest, IL, USA) for the exposure assays. Before the exposures, size distribution and zeta potential of PNPs were certified by a Malvern Zetasizer Nano ZS instrument (Malvern Panalytical Ltd, Malvern, UK; see Supplementary Materials). The nanoparticles' characterization showed some differences in terms of size (Table S1), which were not relevant to the purposes of our study (both PNPs did not exceed the nanosize range).
5
Fluorescent Bead Imaging Protocol
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1- , 1% solid fluorescent bead samples (L2778 fluorescent red, L1030 fluorescent yellow-green, L9654 fluorescent orange, Sigma-Aldrich; Dragon Green, Flash Red, Suncoast Yellow, Bangs Laboratories, Inc.) were diluted 1000-fold in distilled water, then further diluted another 100-fold in 1% low-melt agarose. The 10,000-fold diluted fluorescent bead sample in 1% low-melt agarose was pulled into a glass capillary (5-000-1025, Drummond Wiretrol) with a stainless-steel plunger. After the agarose solidified at room temperature at 20 for 1-2 min, the capillary was transferred to a distilled water-filled imaging chamber and a length of the agarose containing the beads was extruded from the micropipettes allowing optical access.
6
Reducing Fluorescence Photobleaching via Higher Scan Frequencies
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Next, we measured photobleaching rates at 40k and 400k line/s scan frequencies. For this experiment, fluorescent beads (Flash Red, FSFR005, Bangs Laboratories) were continuously imaged over a 100-s interval to produce obvious bleaching. The average laser power under the OL was set to 13.6 mW for both frequencies. Normalized curves of signal intensity during photobleaching showed higher photobleaching under 40 kHz scanning than 400 kHz [Fig. 1(e) ]. Notably, the rate of photobleaching decreased with higher line scan frequency. Comparison of bleaching rates based on time until fluorescence intensity decay to 1/e (36.8%) showed that, at 40 kHz, this decay time was 28.8 s, but 45.8 s at 400 kHz, indicating that signal intensity persisted longer at the higher frequency.
These results were consistent with previous studies20 (link),33 (link) that showed the formation of triplet states is increased under a prolonged, single, continuous illumination time, which ultimately accelerates the photobleaching rate. Taken together, these assays demonstrate that increasing line scan frequency to hundreds of kilohertz can be beneficial for reducing photobleaching rates.
These results were consistent with previous studies20 (link),33 (link) that showed the formation of triplet states is increased under a prolonged, single, continuous illumination time, which ultimately accelerates the photobleaching rate. Taken together, these assays demonstrate that increasing line scan frequency to hundreds of kilohertz can be beneficial for reducing photobleaching rates.
7
In Vivo Tracking of Iron-Oxide Nanoparticles in Mice
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We used 8- to 11-week-old female C57Bl/6 mice (n = 39; Charles River Laboratories, Wilmington, Massachusetts) housed in pathogen-free conditions until use. The SPIO agent Feridex® was used (average diameter, 80-120 nm; Advanced Magnetic Industries, Massachusetts). The 2 fluorescently tagged MPIO particles used were Flash Red and Dragon Green (Bangs Laboratories Inc., Fishers, Indianapolis); their emission and excitation spectra are 620/690 and 500/580, respectively, and the average particle size was 0.9 μm.
All mice in this study were injected IV with either SPIO or MPIO particles at 30 mg of Fe/kg body weight via the tail vein in a total volume of 200 μL of saline or saline only (controls). Mice in group 1 were injected with either SPIO (n = 8) or MPIO particles (Flash Red Bangs Beads; n = 8). Iron-administered mice plus 4 control mice were subject to a prescan and imaged at 1 hour, 24 hours and 1 week after injection.
Mice in group 2 were injected with SPIO (n = 4), MPIO (n = 4), or saline (n = 2), and then sacrificed 24 hours after injection. Femurs and tibias were removed for histological analysis.
Mice in group 3 were injected with MPIO particles (Dragon Green Bangs Beads; n = 7) or saline (n = 2). Animals were sacrificed 24 hours after injection; the splenocytes and bone marrow cells were collected and analyzed for MPIO particle uptake using flow cytometry.
All mice in this study were injected IV with either SPIO or MPIO particles at 30 mg of Fe/kg body weight via the tail vein in a total volume of 200 μL of saline or saline only (controls). Mice in group 1 were injected with either SPIO (n = 8) or MPIO particles (Flash Red Bangs Beads; n = 8). Iron-administered mice plus 4 control mice were subject to a prescan and imaged at 1 hour, 24 hours and 1 week after injection.
Mice in group 2 were injected with SPIO (n = 4), MPIO (n = 4), or saline (n = 2), and then sacrificed 24 hours after injection. Femurs and tibias were removed for histological analysis.
Mice in group 3 were injected with MPIO particles (Dragon Green Bangs Beads; n = 7) or saline (n = 2). Animals were sacrificed 24 hours after injection; the splenocytes and bone marrow cells were collected and analyzed for MPIO particle uptake using flow cytometry.
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