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7 protocols using s8032

1

Viability and Surface Protein Analysis

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Cells were centrifuged and stained for 30 mins at RT with Live/Dead UV Blue stain (Life Technologies, # L23105; diluted 1:1000 in PBS) for assessing viability. After washing, cells were exposed to Fc receptor block (50 μg of unconjugated human IgG; Sigma Aldrich #S-8032) and stained for surface proteins (30 mins at 4°C) by adding respective antibodies in FACs buffer. The FACS buffer is Ca+2/Mg+2-free PBS with 1% heat-inactivated fetal bovine serum (Sigma Aldrich #F2442) and 0.02% sodium azide (Sigma Aldrich #S-8032). For analyzing intracellular proteins, the cells were then subjected to fixation and permeabilization according to the manufacturer’s guidelines (eBioscience, San Diego CA, USA, #00-5123-43, #00-5223-56 and #00-8333-56; BD Biosciences #51-2090KZ, #51-2091KZ) followed by staining with appropriate antibodies. Flow cytometry data was acquired on Fortessa (BD Bioscience) and analyzed with FACSDiva software (BD Biosciences) or Flowjo.
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2

Inhibition of Glycation-Induced Fluorescence

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Bovine serum albumin (BSA; Roche Diagnostics, Basel, Swiss) in a phosphate buffer containing sodium azide (s-8032, Sigma-Aldrich, St. Louis, MO, USA) was added to a 0.2 M solution of glucose and fructose. This solution was added to the OSSCE or aminoguanidine (AG; 396494; Sigma-Aldrich), a positive control. Following 14 days of incubation, the AGE-specific fluorescence was analysed using a spectrofluorometer (Synergy HT; BIO-TEK, Winooski, VT, USA; 370 nm/440 nm). The IC50 (inhibitory concentration which nonenzymatic AGE formation is reduced by half) was calculated from the dose inhibition curve.
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3

Measuring Cellular Proliferation with EdU

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Thymidine is interchangeable with 5-ethynyl-2′-deoxyuridine (EdU) and can thus be incorporated into DNA instead of thymidine in the replication process. This can then be used to measure cellular proliferation by detecting how much EdU was inserted into the DNA. Cells were cultured at a cell seeding density of 1 × 105 and incubated for 24 h with fluorescent azide. Following incubation, the iADSCs were labelled with immunofluorescent markers. A 10% solution of paraformaldehyde (P6148, Sigma-Aldrich) was used to fix cells for 10 min. A 2% BSA solution was used to permeabilize cells for 30 min at room temperature (RT); cells were subsequently washed with PBS three times. Cells were then treated with (1 : 200) α actin primary antibody and incubated at RT for 30 min. The treated iADSCs were rinsed three times with ice-cold washing buffer (azide/PBS/BSA: 0.01% w/v sodium azide (Sigma-Aldrich, S8032)), PBS, and 0.1% w/v BSA (Sigma-Aldrich, A2153) three times. Then, cells were incubated with donkey anti-mouse Alexa fluor 594 secondary antibodies (1 : 800) at RT for 30 min. Cells were again treated with washing buffer and counterstained with 300 nm 4′,6-diamidino-2-phenylindole (DAPI). The counterstained cells were mounted with Fluoromount™ onto microscopic glass cover slides and observed with an Axio Observer Z1 (Carl Zeiss) microscope.
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4

Immunofluorescence Analysis of Cochlear Structures

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Isolated cochleae were immersed in 4% paraformaldehyde and then decalcified with 0.5 M EDTA (Solarbio, E1170). For cryosectioning, cochleae were immersed in increasing concentrations of 10–30% (w/v) sucrose (Biosharp, Amresco 0335) and then with serial mixtures of OCT (Sakura, Tissue-Tek 4583) and sucrose. The sections and whole mounts were blocked with phosphate-buffered saline blocking solution containing 5% donkey serum, 1% bovine serum albumin, 0.02% sodium azide (Sigma-Aldrich, S8032), and 0.5% Triton; incubated with diluted primary antibodies; and further with fluorescence-conjugated secondary antibodies (Alexa Fluor 488/555/647, Invitrogen). The primary antibodies were Atg7 rabbit polyclonal antibody (Thermo Fisher, PA5-35203, 1:300); Myosin VIIa mouse polyclonal antibody (Thermo Fisher, PA1-936, 1:500); Prestin goat polyclonal antibody (Santa Cruz, sc-22694, 1:200); CtBP2 mouse monoclonal antibody (BD Biosciences, 612044, 1:200); and P62 Guinea Pig polyclonal antibody (Progen, GP62-C, 1:200). The anti-fade Fluoromount-G mounting medium (SouthernBiotech, 0100-01) was used for mounting. The fluorescence images were obtained by a Zeiss LSM 710 confocal microscope. For hematoxylin staining, the whole mounts were stained with diluted hematoxylin (Solarbio, G1080) for 5 min.
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5

High-Throughput Cell Staining Protocol

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This protocol was used for staining cells in a 96-well (VWR #82050-748) format and a shaker was used during antibody incubations to obtain uniform staining. The cells were washed twice with 1x PBS (100 µl) and fixed with 75 µl of 4% paraformaldehyde (VWR #AA43368-9M) per well (15 min at RT). Cells were washed with PBS and 75 µl of Blocking Buffer (5% Goat Serum Life Technologies #PCN5000 with 0.32% Triton X-100 Sigma #T8787 in 1× PBS) added per well (1 h at RT). After PBS wash, 50 μl/well of primary antibody (at recommended dilutions in Ab protocol) in the Antibody Buffer (1% BSA and 0.3% Triton X-100 in 1x PBS) was added and incubated at RT, shaking gently in an orbital shaker for 1–2 h for same day processing or overnight at 4 °C while shaking. Cells were washed with PBS and 50 μl/well of secondary antibody in Antibody Buffer (at recommended dilutions in Ab protocol) was added with incubation at RT, shaking gently in an orbital shaker for 1–2 h. After final wash in 1x PBS, plates were wrapped in aluminum foil and stored at 4 °C with 100 µl 0.05% Sodium Azide (NaN3, Sigma #S8032) in PBS.
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6

MOPV Infection Quantification in A549 Cells

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A549 cells were transfected with non-targeting siRNA or ITCH targeting siRNA at a final concentration of 30 nM for 72 h before infection with recombinant MOPV-WT or MOPV-ZFLAG at a MOI of 2 for 24 h. Cells were then harvested, washed, and the pellets suspended 20 min in PBS with 4% formaldehyde. Cells were then permeabilized with PBS complemented with 5% FBS, 0.5% saponin (47036-50G-G, Sigma), 0.02% sodium azide (S-8032, Sigma) before being incubated with anti-FLAG-APC antibody (130-101-565, Miltenyi Biotec, Paris, France) for 30 min at 4 °C. The fluorescence of formaldehyde-fixed cells was measured using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using Kaluza software (Beckman Coulter, version 1.2).
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7

Antibody-DNA Conjugation for Single-Cell Analysis

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Purified, carrier-free monoclonal and polyclonal antibodies (table S2) were conjugated to unique DNA oligonucleotides (TriLink BioTechnologies) as described before (28 (link)). Each antibody was concentrated on a preblocked 50-kDa centrifugal filter column (Amicon Ultra, EMD Millipore, no. UFC505096), and a partial antibody reduction was performed using a Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)reduction solution containing 2.5 mM TCEP (Sigma-Aldrich, no. C4706-10G) and 2.5 mM EDTA (Sigma-Aldrich, no. 93302) in phosphate-buffered saline (PBS; pH 7.0). The partial reduction was allowed to run for 30 min. Toluene-deprotected, lyophilized, and maleimide-modified DNA oligonucleotides were then conjugated to the antibodies at a 2:1 w/w ratio for 2 hours, with at least 100 μg of antibody per reaction. Conjugated antibodies were washed and eluted in PBS-based antibody stabilizer (Thermo Fisher Scientific, no. NC0436689) containing 500 mM NaCl, 5 mM EDTA, and 0.1% (w/v) NaN3 (Sigma-Aldrich, no. S8032).
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