In total, 266-6 cells were seeded at a density of 50,000 cells per well of a 6-well plate. Twenty-four hours later, cells were transduced with lentiviral supernatants supplemented with protamine sulphate (10 µg/mL, Sigma-Aldrich) for 48 h. After splitting, cells were selected for at least 1 week in 0.4 µg/mL puromycin (Invitrogen) before any further analysis was performed. Human exocrine cells were transduced with lentiviral shRNA-Evi1 vectors (MOI 10) on the day of isolation for 16 h at a density of 200,000 cells in advanced RPMI supplemented with 5% heat-inactivated FBS, 1% GlutaMax, and 1% Pen/Strep supplemented with protamine sulphate (10 µg/mL, Sigma-Aldrich). Cells were then washed with PBS and further cultured for 4–7 days in Advanced RPMI supplemented with 5% heat-inactivated FBS, 1% GlutaMax, and 1% Pen/Strep.
Protamine sulphate
Manufactured by Merck Group
Sourced in United States
Protamine sulphate is a laboratory reagent used in various applications. It is a naturally occurring protein derived from fish sperm. Protamine sulphate is commonly used as an anticoagulant reversal agent in medical and research settings.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamin 2000, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (2 mentions)
Fugene 6, by Promega (2 mentions)
Pen strep, by Merck Group (1 mentions)
Glutamax, by Merck Group (1 mentions)
Maxisorp immunoplate, by Thermo Fisher Scientific (1 mentions)
Goat anti human igg hrp, by Thermo Fisher Scientific (1 mentions)
Human igg elisa quantitation kit, by Fortis Life Sciences (1 mentions)
Opteia tmb substrate, by BD (1 mentions)
Goat anti mouse igg hrp, by Southern Biotech (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Optima l 100 xp, by Beckman Coulter (1 mentions)
Calcium phosphate, by Merck Group (1 mentions)
Puromycin aminonucleoside, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (1 mentions)
Macrophage colony stimulating factor, by Thermo Fisher Scientific (1 mentions)
Interleukin 2 (il 2), by Merck Group (1 mentions)
23 protocols using protamine sulphate
1
Lentiviral Transduction of Exocrine Cells
2
Quantifying Anti-dsDNA IgG Autoantibodies in Lupus
3
Lentiviral Transduction of mMSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mMSCs were seeded (6 × 105 cells/flask) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine at 37 °C with 5% CO2. mMSCs were transduced with recombinant lentivirus (LV-PUUV-S, LV-PUUV-M, and LV-Katushka2S) carrying PUUV N, Gn/Gc, or red fluorescent protein Katushka2S (MOI 100) using Opti-MEM medium (Gibco, Grand Island, New York, USA) supplemented with 8 µg/mL protamine sulphate (Sigma, Saint Louis, USA). Fresh media was added after 24 h to support cell proliferation. The medium and cells were collected 48 h later.
4
Lentiviral Transduction of Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines were transduced in 24-well plates by adding concentrated viral supernatant to 2 × 105 cells in 500 μl medium in the presence of protamine sulphate (6 μg ml−1) and spinoculation (1000 × g, 1 h, 32°C). Transduction of murine lin− cells isolated from bone marrow cells was done with the same protocol after 24 h prestimulation at a multiplicity of infection of 20–50.
For transduction, 1 × 105 human or murine PSC were seeded as single cells onto Matrigel- (Beckton & Dickinson, Heidelberg, Germany) or gelatine-coated 12-well plates in standard medium containing, respectively. The next day, cells were transduced with lentiviral vectors (MOI 10–50) in the presence of 10 μg ml−1 protamine sulphate (Sigma Aldrich). After 3–4 days cells were transferred to MEF cells and cultured as described above.
For transduction, 1 × 105 human or murine PSC were seeded as single cells onto Matrigel- (Beckton & Dickinson, Heidelberg, Germany) or gelatine-coated 12-well plates in standard medium containing, respectively. The next day, cells were transduced with lentiviral vectors (MOI 10–50) in the presence of 10 μg ml−1 protamine sulphate (Sigma Aldrich). After 3–4 days cells were transferred to MEF cells and cultured as described above.
5
Lentiviral Transduction of SOX2 Reporter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A SOX2 pluripotency response reporter construct containing a SOX2 response element upstream of a minimal CMV promotor driving copGFP and luciferase was purchased from BioCat GmbH, Heidelberg, Germany as pre-packaged lentiviral particles. For lentiviral infection, T47D cells were seeded into six-well plates and incubated with lentiviral particles at a multiplicity of infection (MOI) of 1.5 in the presence of 5 μL/mL protamine sulphate (Sigma-Aldrich, St. Louis, MO, USA). The lentivirus solution was replaced with normal media 24 h later. Cells were cultured for 3 days to await the development of GFP signal at which point they were passaged and selected using puromycin.
6
Silencing β2-microglobulin in Hepatocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A lentiviral vector system was used to deliver short hairpin RNA (shRNA) to silence β2‐microglobulin (shβ2m) expression. A vector encoding for a non‐sense sequence was used as a control shRNA (shNS). The vector was produced as previously described.8 Briefly, HEK293T cells were cotransfected with psPAX2, pMD2.G and the vector encoding for the shRNA sequence using calcium phosphate (Sigma‐Aldrich, Steinheim, Germany). Lentiviral vector containing supernatants were harvested 48 hours after transfection, filtered and concentrated by ultracentrifugation at 30 000 g (Optima L‐100 XP, Beckmann Coulter, Krefeld, Germany), for 4 hours. The pellets containing the viral vectors were resuspended in Williams's Medium. For hepatocyte transduction, 1.5 × 106 cells were seeded in a 6‐well plate and infected overnight with the vectors in the presence of 8 µg/mL protamine sulphate (Sigma‐Aldrich). Afterwards, hepatocytes were washed and cultured with fresh culture medium.
7
Podocyte Injury and Growth Factor Responses
8
Lentiviral Reprogramming Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lentiviral particle production was performed as described previously [63] (link). A 4-in-1 reprogramming vector harboring 2A-linked hOct4, hKlf4, hSox2 and hc-Myc and an IRES-linked dTomato (hOKSM.idTomato) was used [26] (link). To determine biological titers, human HT1080 fibroblasts were transduced with viral supernatants and expression of virally delivered fluorescent protein dTOMATO was measured by flow cytometry 4 days post transduction (p.t.). Titers were calculated as follows: [(cell number at transduction) x (frequency of transduced cells) x 2]/(volume of viral supernatant). Viral transductions were performed in presence of 10 mM HEPES and 4 µg/ml protamine sulphate (Sigma) for 8–16 h.
9
Recombinant Soluble β3-Integrin Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Soluble β3-integrin was produced recombinantly in HEK293 as previously described [19] (link). The recombinant sβ3-integrin sequence was designed based on the RNA sequence detected in the AML patients showing increased serum levels of sβ3-integrin. Therefore, the recombinant soluble β3-integrin is expected to have the same effect as the protein found in the AML patient’s serum. Briefly, a lentiviral vector encoding the sequence for sβ3-integrin was used for the HEK293 cells transduction in the presence of 8 µg/mL protamine sulphate (Sigma Aldrich Chemie, Munich, Germany). Seventy-two hours after transduction, cell culture supernatant was analyzed by ELISA for the presence of sβ3-integrin as previously described [19] (link). sβ3-integrin producing HEK 293 cells were cultured in bioreactors (CELLine Adherent, Integra, Fernwald, Germany) to increase the protein production yields. Cell-culture supernatants were harvested and stored at −20°C until the purification step.
10
Bmi1 Retroviral Overexpression Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pMSCV-neo retroviral vector (MSCV) was used to construct the recombinant transfection vector containing the full-length cDNA of human Bmi1, which was cloned from K562 with a confirmed correct sequence. The plasmid was transfected into the PT67 package cell line (Clontech, Mountain View, CA, USA) using a 3:1 ratio of lipofectamin 2000 (Invitrogen) versus DNA. Six hrs after transfection, the transfection reagent was removed and replaced by IMDM (Invitrogen) with 20% FBS (Invitrogen). Cells were re-fed after 24 hrs in complete media plus 1 g/l G418 (Amresco, Solon, OH, USA). Supernatants were pooled and filtered through 0.45 μm syringe filter for immediate use. U937 and K562 were cultured in supernatant containing retroviral particles in presence of 0.6 g/l protamine sulphate (Sigma-Aldrich, St. Louis, MO, USA). Twenty-four hrs after infection, cells were moved to complete media plus 0.5 g/l G418. Subclones of Bmi1-transfected cell were obtained by limited dilution.
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!