To perform surface staining, 1 × 106 cells were placed in individual wells of a 96-well round bottom plate and incubated with the appropriate antibody cocktails for 15 min at 4°C on a slow rocker. After the staining, cells were fixed in a solution of 2% ultrapure formaldehyde (Polysciences, Inc., Warrington, PA, USA) in FACS buffer for 20 min on ice, washed twice, and analyzed the following day on the Canto II (BD Biosciences) or FACSCalibur (BD Biosciences). Intracellular staining was performed using Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with BD GolgiStop (BD biosciences) according to the manufacturer’s instruction. Flow cytometry acquisition was performed on an LSRIISorp. Data were analyzed using FACS Express or FlowJo software (Tree Star, Inc., Ashland, OR, USA). Antibodies against CD45 (clone 30-F11, BD Pharmingen), CD3 (clone 145-2C11, BD Pharmingen), CD4 (clone GK1.5, BD Pharmingen), CD8 (clone 5H10, Biolegend), T-bet (clone eBio4B10, eBioscience), IL-17A (clone ebio17B7, eBioscience), IL-4 (clone B11B, Biolegend), IFNγ (clone XMG 1.2, eBioscience), IL-22 (clone A3.6M, eBioscience and clone poly5164, Biolegend), TGF-β (clone 11A5, Biolegend), IL-17F (clone ebio18F10, eBioscience), NKP46 (clone 29A 1.4, Biolegend), c-Kit (clone 2B8, Biolegend), Sca-1 (clone D7, BD Pharmingen), and CD127 (clone A7R34, eBioscience) were used.
Ultrapure formaldehyde
Manufactured by Polysciences
Sourced in United States
Ultrapure formaldehyde is a high-purity, laboratory-grade chemical compound. It serves as a core component in various research and analytical applications that require a reliable and consistent source of formaldehyde.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospo p44 42 mapk erk1 2 clone e10, by Cell Signaling Technology (2 mentions)
Cd45 clone 30 f11, by BD (1 mentions)
Cd3 clone 145 2c11, by BD (1 mentions)
Cd4 clone gk1, by BD (1 mentions)
T bet clone ebio4b10, by Thermo Fisher Scientific (1 mentions)
Il 17a clone ebio17b7, by Thermo Fisher Scientific (1 mentions)
Ifn γ clone xmg1, by Thermo Fisher Scientific (1 mentions)
Nkp46 clone 29a1, by BioLegend (1 mentions)
Sca 1 clone d7, by BD (1 mentions)
Cd127 clone a7r34, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Canto 2, by BD (1 mentions)
α mem, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Acetone, by Thermo Fisher Scientific (1 mentions)
Anti γh2ax, by Merck Group (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
Digital microphotography system dfx1200, by Nikon (1 mentions)
Act 1, by Nikon (1 mentions)
7 protocols using ultrapure formaldehyde
1
Multiparametric Flow Cytometry Analysis
2
Phosphorylation of Syk and ERK in moDCs
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For detection of phosphorylation of Syk (pSyk) and ERK (pERK), 2.5 × 104 immature moDCs were seeded overnight in a 96-well flat-bottom plate. moDCs were stimulated with SEA (50 μg/ml), SEAΔα-1/ω-1 (50 μg/mL), or ω-1 (500 ng/mL) in the presence or absence of blocking antibodies or inhibitors (R406, anti-MR, anti–Dectin-1, anti–Dectin-2, combination of anti–Dectin-1 and anti–Dectin-2 or IgG1 and IgG2 control antibodies) for indicated periods, and the moDCs were fixed for 15 min with 4% ultrapure formaldehyde (Polysciences, Warrington, PA) directly in the plate. The cells were harvested and washed first with PBS and then with 0.5% of saponin for permeabilization. Cell were intracellularly stained with anti–phospo-Try525/526 Syk (clone C87C1) and anti–phospo-p44/42 MAPK (Erk1/2) (clone E10) (both Cell Signalling Technology). Following 2-h incubation at room temperature, cells were washed with 0.5% of saponin, and Syk and ERK phosphorylation was determined by flow cytometry.
3
Primary Bone Marrow Macrophage Isolation
4
Evaluating γH2AX Foci in SV-HUC Cells
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SV-HUC cells were treated with BCEVs (40 µg/mL) for 18 h in chamber slides. Cells were then washed with PBS and fixed in 1% ultrapure formaldehyde (catalog no. 04018, Polysciences) in PBS for 20 min at room temperature. After additional washing with PBS, cells were treated with methanol for 10 min at −20 °C, air dried, and permeabilized with acetone (catalog no. A18-1, Fisher Scientific) for 1 min at room temperature. Cells were then blocked with 5% normal swine serum for 1 h at room temperature prior to overnight primary antibody incubation at 4 °C using anti-γH2AX (1:400; catalog no. 05-636, Millipore) and secondary staining with Alexa Fluor 488 F(ab’)2 fragment IgG (catalog no. A11017, Invitrogen). DAPI was used for nuclear counterstaining. γH2AX foci and nuclei were counted using an ImageJ plugin (EZ foci PZ).
5
Quantifying pERK in moDCs
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For the detection of ERK phosphorylation (pERK), 2.5 × 104 immature moDCs were seeded overnight in a 96-well flat-bottom plate. moDCs were stimulated with SEA (25 μg/mL) and Δω1-SEA (25 μg/mL) for the indicated periods, and the moDCs were fixed for 15 min with 4% ultrapure formaldehyde (Polysciences, Warrington, PA, USA) directly in the plate. The cells were harvested and washed first with PBS and then with 0.5% of saponin for permeabilization. The cells were intracellularly stained with anti-phospo-p44/42 MAPK (Erk1/2) (clone E10) (both Cell Signalling Technology). Following 2 h of incubation at room temperature, the cells were washed with 0.5% of saponin, and ERK phosphorylation was determined by flow cytometry.
6
Dynamin Regulation of HSV-1 Infection
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WT HA‐dynamin 2 pcDNA3.1 (34684) and K44A HA‐dynamin 2 pcDNA3.1 (34685) were obtained from Addgene. AP180‐C myc and VPS4‐EQ YFP were described previously 24 , 54 . Plasmids of interest or empty pcDNA3.1 were co‐transfected with pcDNA‐UL3625 or empty control plasmid into COS7 cells. Next day cells were infected at multiplicity of infection (MOI ) of 5 PFU/mL with HSV‐1ΔUL36 virus. After 16‐h infection cells and supernatants were harvested together and prepared for titration by three freeze–thaw cycles. Virus titers were assessed using the pUL36 complementing cell line HS30. Cells for WB analysis were lysed with 1% Triton‐X‐100 in PBS with protease inhibitors cocktail (Roche) and run on SDS–PAGE followed by detection of VP16 and actin. Cells for immunostaining were fixed with 4% ultra‐pure formaldehyde (Polysciences, cat # 04018‐1) 10 h after infection. Antibodies specific to gD (LP2) were added 15 min before fixing. For transferrin uptake cells were transfected with pcDNA3.1, dominant negative AP180, dynamin WT or dynamin K44A for 24 h. Cells were incubated with Transferrin Alexa Fluor 568 for 5 min in serum‐free medium. After fixing and permeabilization immunodetection of HA‐tag and Myc‐tag was performed.
7
Lung Tissue Collection and Preparation
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