Cytoplasmic and nuclear extracts were prepared as described previously (Bélanger et al, 2010 (link)). Briefly, A549 cells with or without overnight IFN‐β 1a (PBL Assay Science, 11410‐2) treatment (1,000 U/ml) were detached from cell culture dish and resuspended in 600 μl of cold lysis buffer (10 mM HEPES (pH 7.5), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 1 × Protease/phosphatase Inhibitor Cocktail (Cell Signaling Technology, 5872S)). The cell suspension was incubated on ice for 15 min before addition of NP‐40 to the final concentration of 0.5% and 10‐s vortexing. The resulting mixture was centrifuged at 2,000 × g for 30 s at 4°C before the supernatant (cytoplasmic fraction) was removed. The pellet was resuspended in 100 μl of nuclear extraction buffer (20 mM HEPES (pH7.5), 400 mM KCl, 1 mM DTT, and 1 × Protease/phosphatase Inhibitor Cocktail), incubated on a rotating wheel at 4°C for 15 min, and centrifuged at 14,000 × g for 15 min at 4°C. The supernatant (nuclear fraction) was further harvested and frozen at −20°C until further use.
Ifn β1a
Manufactured by PBL Assay Science
Sourced in United States, Macao
IFN-β1a is a recombinant human interferon beta-1a protein. It is produced using mammalian cell culture technology. IFN-β1a is used in research applications to study the biological properties and functions of interferon beta.
Automatically generated - may contain errors
Lab products found in correlation
Sigma tr 1003 g, by Merck Group (2 mentions)
Ifn α2a, by PBL Assay Science (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Ifn γ, by PBL Assay Science (1 mentions)
Genejuice reagent, by Merck Group (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Mouse ifn β, by PBL Assay Science (1 mentions)
Ifn γ, by Merck Group (1 mentions)
Minocycline hydrochloride, by Merck Group (1 mentions)
Anti cd3 coated 96 well plates, by BD (1 mentions)
Dynabeads flowcomp human cd4 kit, by Thermo Fisher Scientific (1 mentions)
N ethylmaleimide nem, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Benzonase, by Merck Group (1 mentions)
8 protocols using ifn β1a
1
Preparation of Cytoplasmic and Nuclear Extracts
2
IFN-β1a Pre-treatment of Nucleofected CD4+ T Cells
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Nucleofected CD4+ T cells were treated with 1000 U/mL IFN-β1a (PBL Assay Science #11410) 24 h prior to HIV infection. For the first set of single KO experiments (Figure 2 ) and bulk RNA-seq experiments (Figure 3 ), cells were IFN-treated six days post nucleofection. Otherwise, cells were IFN-treated nine days post nucleofection to allow for additional recovery. For targeted KO experiments, half of each nucleofection was IFN-treated, whereas all cells were IFN-treated for CRISPR screens. For ISG KO spreading infections, cells were resuspended to 1e6 cells/mL in RPMI complete media with 100 U/mL IL-2 and 100 μL was added in duplicate for each virus to 96-well flat-bottom plates. Cells were infected with 10 μL of virus at an MOI of 0.02 in the presence of 8 μg/mL polybrene (Millipore Sigma #TR-1003-G) by spinoculation at 1100×g for 90 min at 30C. After infection, cells were transferred to 96-well V-bottom plates, pelleted at 300×g for 10 min, and resuspended in 100 μL of fresh RPMI complete with 100 U/mL IL-2 that contained 1000 U/mL IFN as necessary. Cells were then transferred back to the 96-well flat-bottom infection plates and were incubated at 37C. Viral supernatants were harvested 2, 4, 6, and 8 days post-infection (dpi), stored at −80C, and replaced with RPMI complete media with 100 U/mL IL-2 containing 1000 U/mL IFN as needed.
3
Cell culture and transfection protocol
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HEK293T and A549 cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 100 U/ml of penicillin/streptomycin (Thermo Fisher Scientific, MA, USA) in humidified chamber at 37 °C, supplemented with 5% CO2. HEK293T cells were transfected with Genejuice reagents (MilliporeSigma, MA, USA). A549 cells were transfected by use of Lipofectamine 3000 (Thermo Fisher Scientific). IFN-β1a and IFN-γ were purchased from PBL Assay Science (NJ, USA).
4
Analytical Specificity of Simoa IFN-α Assay
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To verify the analytical specificity of the Simoa IFN-α Advantage Kit and HEK293-3C11-ISRE reporter cells, serum from an HC or special stripped serum (Valley Biomedical, Winchester, VA, USA) were spiked with recombinant human IFN-α1(D), -D(1), -A(2a), -2(2b), -4a(M1), -4b(4), -G(5), -K(6), -J1(7), -B2(8), -H2(14), -WA(16), -I(17), -F(21), IFN-β1a (all from PBL Assay Science, Tebu-bio, Heerhugowaard, The Netherlands) or IFN-γ (PeproTech, Cranbury, NJ, USA) and snap frozen or assayed directly.
5
IFN-β1a Treatment of Nucleofected CD4+ T Cells
6
Interferon Antiviral Activity Assay
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Vero and A549 cells were seeded into 96-well plates for 24 h and treated with human IFN-α-2b (Intron A, Schering Corporation, NJ), IFN-β1a (PBL, NJ) or IFN-γ (Sigma-Aldrich, MO) at 125, 250, 500 and 1000 U/ml for 16 h. MEF cells were treated with mouse IFN-β (PBL) as indicated. Cells were then infected with VSV, Candid#1 JUNV or Romero JUNV at an MOI of 0.1 PFU/cell. IFNs were supplemented after virus infection. For Romero and Candid#1 JUNV infection, supernatants were collected at 3 days post infection and assayed for virus production by plaque assay. For VSV infection, supernatants were collected at 16 hr p.i.. Data represent the mean of three experiments ±SEM.
7
Minocycline Modulation of CD4+ T Cell Activation
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CD4+ T cells were isolated from PBMCs by the Dynabeads FlowComp Human CD4 kit (Invitrogen). Purity was consistently >95% of live cells as determined by CD3 and CD4 staining by flow cytometry. CD4+ T cells were grown in 96-well plates or anti-CD3 coated 96-well plates (BD) with or without 20 μM minocycline hydrochloride (Sigma) in 100 μl RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM HEPES buffer, and 2 mg/mL gentamicin. After 24 hours, a 50 μL aliquot containing 60 μM minocycline was added to replenish minocycline at a final concentration of 20 μM in the 150 μL well volume. Some wells were also stimulated with 1,000 U/mL each of IFNα-2a and IFNβ-1a (PBL). All wells had a final volume of 150 μL and were cultured for an additional 24 hours (total 48 hours) before flow cytometry analysis.
8
Interferon-Stimulated Protein Profiling
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HEK293, BEAS-2B and HAECs were maintained as described. To induce interferon-stimulated gene proteins that may be 3CLpro substrates, the BEAS-2B cells were cultured in DMEM/F12 with 10% (v/v) FBS and treated with 104 U/ml carrier-free IFN-α2a or IFN-β1a (PBL Assay Science), or medium (control) for 18 h. Cells were harvested, and lysates were prepared under native conditions taking the necessary steps to reduce any cellular proteolytic activity. All steps were performed on ice. First, cells were washed twice with phosphate-buffered saline (PBS) and lifted with Versene buffer (0.5 mM EDTA, PBS). The cell pellet was washed twice with 150 mM NaCl, 50 mM Tris-HCl, pH 7.2 by centrifugation at 300g for 10 min. Protease inhibitor cocktail (bimake.com ) and 5 mM N-ethylmaleimide (NEM) (Sigma) were added to the cell pellet in hypotonic lysis buffer (2 mM MgCl2, 1 μl/ml Benzonase (Sigma), 50 mM Tris-HCl, pH 7.2). For lysis, cells were pushed through a 27-gauge needle for 10 cycles and rested on ice for 1 h with agitations every 10 min. Cell lysates were flash-frozen in liquid N2 and stored at –80°C.
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