HAE cells grown on Transwell filters were incubated with 20 µl of DMEM ± 100 µM specified peptides (Fig. 4a ) on day 28 of establishing ALI. Then, 24 h later, cells were fixed for 20 min in 2% paraformaldehyde in DPBS. Cells were then permeabilized for 10 min with 0.2% saponin and 10% FBS (Thermo Fisher Scientific) in DPBS. Cells were washed twice with DPBS and stained with anti-MUC5AC (45M1, MA1-21907, Thermo Fisher Scientific) antibodies diluted 1:100 in DPBS, 0.2% saponin and 10% FBS overnight at 4 °C. Subsequently, cells were washed twice with DPBS and incubated for 1 h at room temperature in DPBS, 0.2% saponin and 10% FBS containing AlexaFluor-488-labelled anti-mouse secondary antibodies (1:500; Thermo Fisher Scientific) and DAPI (1:5,000; Thermo Fisher Scientific). Images were taken on an inverted confocal microscope (Leica TCS SP5) using a ×40 lens (Leica HC PL APO CS2 40x1.30 OIL). Images for the blue (DAPI), green (AlexaFluor 488) and red (Cy3) channels were taken in sequential mode using appropriate excitation and emission settings.
Saponin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Saponin is a class of natural compounds derived from plants. It is a surfactant that can be used in various laboratory applications, particularly in the field of biochemistry and cell biology. Saponin has the ability to interact with and disrupt cell membranes, which can be utilized for specific research purposes. However, a detailed description of its core function and intended use would require more specific information about the particular application or research context.
Automatically generated - may contain errors
Lab products found in correlation
Saponin, by Merck Group (19 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Tcs sp5, by Leica (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (3 mentions)
Macsquant vyb flow cytometer, by Miltenyi Biotec (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Alexa fluor 488 goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (2 mentions)
0.4 μm filter, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Fluoromount g, by Thermo Fisher Scientific (2 mentions)
Click it cell reaction buffer kit, by Thermo Fisher Scientific (2 mentions)
65 protocols using saponin
1
Immunofluorescence Staining of Airway Epithelial Cells
2
Quantifying RORγ Nuclear Localization
3
Immunocytochemical Staining Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After intoxication, the cells were washed with phosphate-buffered saline (PBS; Pan Biotech) and fixed with a solution of 4% paraformaldehyde (PFA; Sigma Aldrich) in PBS, pH 7.3, for 20 min at room temperature (RT). They were washed twice again in PBS, and were then permeabilized and the non-specific sites blocked using a solution of PBS containing 0.1% of saponin (Sigma Aldrich) and 1% of FCS, for 15 min at RT. All primary antibody (Ab) incubations were performed in PBS containing 1% of FCS, 0.1 mg/mL of saponin, for 2 h, at RT. The cells were then washed with PBS containing 1% of FCS, 0.1% of saponin, and incubated with the appropriate secondary Ab (goat anti-mouse, labeled with Alexa 488 or goat anti-rabbit, labeled with Alexa 568, Invitrogen), used at 5 μg/mL in PBS containing 1% of FCS and 0.1% of saponin, for 1 hour at RT. The different Ab, and detailed procedures used throughout this study are listed in S1 Table .
4
Cardiac Troponin T Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Day 15 cells were dissociated into single cells using 0.2 ml of 0.25% Trypsin (UCSF Cell Culture Facility) per well of a 24-well plate with incubation at 37 °C. Cells were resuspended in 0.8 ml/well EB medium (KO DMEM supplemented with 20% FBS (HyClone), Glutamax (Gibco), non-essential amino acids (UCSF Cell Culture Facility), and 0.1 mM beta-Mercaptoethanol (Sigma)). 200 μL (about 200,000) cells were pelleted and fixed in 4% paraformaldehyde at room temperature for 15 min. Cells were permeabilized in FACS buffer (DPBS without calcium and magnesium, 4% FBS, and 2 mM EDTA (Gibco)) with 0.5% (w/v) saponin (Sigma). Cells were stained with 100 μl of 0.002 μg/μl (1:100) Mouse anti-human cardiac Troponin T (cTnT) primary antibody (Thermo, MS-295-P) in FACS buffer with saponin at room temperature for 30 min and washed. Cells were then stained with 100 μl of 0.01 μg/μl (1:200) Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Invitrogen, A-11029) in FACS Buffer with saponin at room temperature for 30 min and washed. Finally, cells were stained with 100 μl of 0.01 μg/μl (1:1000) Hoechst 33342 (Molecular Probes) in FACS buffer at room temperature for 5 min and passed through a 0.4 μm filter (Millipore) to remove cell clumps. Data were collected using the MACSQuant VYB flow cytometer (Miltenyi Biotec) and analyzed using FlowJo.
5
Cardiac Troponin T Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Day 15 cells were dissociated to single cells using 0.2 mL of 0.25% Trypsin (UCSF Cell Culture Facility) per well of a 24-well plate with incubation at 37°C. Cells were resuspended in 0.8 mL/well EB medium (Knockout DMEM supplemented with 20% FBS (HyClone), Glutamax (Gibco), Non-essential Amino Acids (UCSF Cell Culture Facility), and 0.1 mM beta-Mercaptoethanol (Sigma)). 200 μl (about 200,000) cells were pelleted and fixed in 4% paraformaldehyde at room temperature for 15 minutes. Cells were permeabilized in FACS Buffer (DPBS without calcium and magnesium, 4% FBS, and 2 mM EDTA (Gibco)) with 0.5% (w/v) saponin (Sigma). Cells were stained with 100 μl of 0.002 μg/μl (1:100) Mouse anti-human cardiac Troponin T (cTnT) primary antibody (Thermo, MS-295-P) in FACS Buffer with saponin at room temperature for 30 minutes and washed. Cells were then stained with 100 μl of 0.01 μg/μl (1:200) Alexa Fluor 488 goat anti-mouse IgG secondary antibody (Invitrogen, A-11029) in FACS Buffer with saponin at room temperature for 30 minutes and washed. Finally, cells were stained with 100 μl of 0.01 μg/μl (1:1000) Hoechst 33342 (Molecular Probes) in FACS buffer at room temperature for 5 minutes and passed through a 0.4 μm filter (Millipore) to remove cell clumps. Data were collected using the MACSQuant VYB flow cytometer (Miltenyi Biotec) and analyzed using FlowJo.
6
Visualizing Autophagy in BCG-Infected Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Differentiated THP-1 cells were seeded onto a poly-D-lysine coated, 96-well glass-bottom black tissue culture plate (4.5 × 104 cells/well) and kept in RPMI-1640 medium minus phenol red (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS at 37°C/5% CO2. Cells were infected with BCG at a MOI of 10, with/without 100 nM 7α,25-OHC, with/without 10 µM GSK682753 for 2 h, washed and incubated for a further 4 h with agonists and antagonists. Cells were then fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.05% saponin (Sigma Aldrich) for 20 min and blocked with 1% BSA, 0.05% saponin (Sigma Aldrich) for 1 h. Cells were immunolabeled with rabbit anti-human LC3B (ThermoFisher L10382; 1:1,000), 0.05% saponin at room temperature for 1 h followed by Alexa FluorTM 647 goat anti-rabbit IgG (ThermoFisher A21245; 1:1,000), 0.05% saponin at room temperature for 1 h followed by nuclear staining with Hoechst 33342 (Thermo Fisher Scientific 62249; 1:2,000) for 15 min. Cells were washed, and confocal microscopy was performed using the Olympus FV3000, 60× magnification. Images obtained were analyzed with the ImageJ software (24 (link)).
7
Lymph Node Immune Cell Profiling
8
Immunofluorescence Imaging of Listeria-infected Macrophages
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lm EGDGFP-infected BMDMs were fixed in 3% paraformaldehyde (15 min), quenched with 20 mM NH4Cl (1 h) and blocked with 1% BSA (sigma) in PBS (30 min). Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and labeled with Alexa Fluor 647-conjugated phalloidin (Invitrogen), during 45 min on the dark. Cells were washed and slide preparations were mounted and dried at room temperature. Images were captured with an Olympus BX53 fluorescence microscope. The percentage of cells with intracellular bacteria or beads, and the number of intracellular bacteria or beads per cell were calculated. At least 300 cells were analyzed for each sample in three independent experiments. Non-infected and Lm-infected HUVEC were incubated for 1 h with primary antibody rabbit anti-STAB-1 (1:100, Millipore), diluted in 0.2% saponin (Merck) supplemented with 1% BSA. Cells were washed in 0.2% saponin and incubated 45 min with secondary anti-rabbit Alexa 488 antibody (Invitrogen). DNA was counterstained with DAPI (Sigma) and actin labeled with TRITC-conjugated phalloidin. Images were collected with an Olympus BX53 fluorescence microscope and processed using ImageJ.
9
Kinetic IL-5 Activation and Intracellular Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD300f or empty vector expressing I.29 cells were activated with 100 ng/ml IL-5 (Peprotech, Rehovot, IL) kinetically (0–45 minutes). Subsequently, the cells were fixated with 3.7% formaldehyde and stained with Alexa Fluor 647 conjugated ERK1/2 antibody (Cell Signaling, Danvers, MA) in saponin (Invitrogen, Grand Island, NY) supplemented buffer for intracellular staining. IL-5Rα expression was determined using Alexa Fluor 488 conjugated IL-5Ra antibody (BD Bioscience, San Jose, CA).
10
Phosphorylation Kinetics in RKC-2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RKC-2 cells were starved for 1 h and aliquoted to Eppendorf tubes (106 cells/ 90 µl RPMI 1640). They were then incubated (37 °C, 5 min) followed by activation with 10 U/ml EPO for 0, 10, and 30 min. The reaction was stopped with 10 µL of 37% formalin, giving a final concentration of 3.7% formalin. Cells were incubated for 1 min at 37 °C, and then cooled on ice for 5 min and centrifuged (3,000 rpm for 2 min). After this, the cells were washed once with 1% BSA/PBS and centrifuged (3,000 rpm for 2 min). Staining for the intracellular proteins [(PE eFluor 610 pSTAT5 (eBioscience 61-9010-42) and p-ERK (sc-7383) followed by an FITC conjugated Goat anti-mouse antibody] was done in Saponin (Invitrogen) 1%BSA/PBS. The cells were incubated for 1 h in the dark at room temperature and then centrifuged and resuspended in Flow buffer. The stained cells were examined by GalliosTM Flow cytometer and analyzed using Kaluza Analysis Software (Beckman Coulter, Nyon Switzerland).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!