HeLa cells, HEK 293T cells, and the HIV-1 indicator cell lines (LC5-RIC and LC5-RIC-R5) were kept under standard conditions at 37°C in 5% CO2 in Dulbecco’s modified Eagle medium (DMEM containing GlutaMAX-1; Gibco, Darmstadt, Germany) or very-low-endotoxin(VLE)-RPMI 1640 medium (Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (Biochrom AG) and 1% antibiotic-antimycotic solution (Gibco). In order to maintain stable reporter and CD4 receptor expression of LC5-RIC/LC5-RIC-R5 reporter cells, 0.74 mg ml−1 Geneticin (G418 sulfate; PAA Laboratories, Pasching, Austria) and 0.13 mg ml−1 hygromycin B (PAA Laboratories) were added to the cell culture medium at every second passage.
Hygromycin b
Manufactured by GE Healthcare
Sourced in Austria, United Kingdom
Hygromycin B is a laboratory reagent used as a selectable marker in genetic engineering applications. It is an antibiotic that inhibits protein synthesis in eukaryotic cells, allowing for the selection of cells that have been successfully transfected or transformed with a gene of interest.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax 1, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Vle rpmi 1640 medium, by Harvard Bioscience (1 mentions)
Geneticin g418 sulfate, by GE Healthcare (1 mentions)
Y 27632, by Merck Group (1 mentions)
Rac1 inhibitor, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Gm6001, by Merck Group (1 mentions)
Blebbistatin, by Merck Group (1 mentions)
Foetal calf serum, by GE Healthcare (1 mentions)
Gapdh, by Abcam (1 mentions)
General reagents, by Merck Group (1 mentions)
H1152, by Merck Group (1 mentions)
On targetplus smartpool sirna, by Horizon Discovery (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
Neomycin, by Thermo Fisher Scientific (1 mentions)
Ficoll, by Merck Group (1 mentions)
3 protocols using hygromycin b
1
Maintaining HIV-1 Reporter Cell Lines
2
Migration Assay with Cell Signaling Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All tissue culture, inhibitors and general reagents were purchased from Sigma-Aldrich (Poole, UK), except AA (MP Biomedicals, London, UK), which was emulsified at 10 mg ml−1 in methyl-β-cyclodextrin. Hygromycin B and foetal calf serum (FCS) were from PAA Laboratories (Yeovil, UK). Matrigel and 8 μm FluoroBlok cell-culture inserts were from BD Biosciences (Oxford, UK).
Antibodies were from Cell Signalling Technology (New England BioLabs (UK) Ltd, Hitchin, UK) except GAPDH, which was from Abcam (Cambridge, UK). Y-27632, Rac-1 inhibitor, GM6001, H1152 and blebbistatin were from Merck Biosciences (Nottingham, UK). All On-Target Plus Smartpool siRNAs were from Dharmacon, Thermo Fisher Scientific (Fermentas GmbH, St Leon, Germany). Protease and phosphatase inhibitor cocktail were purchased from Roche (Sussex, UK).
Antibodies were from Cell Signalling Technology (New England BioLabs (UK) Ltd, Hitchin, UK) except GAPDH, which was from Abcam (Cambridge, UK). Y-27632, Rac-1 inhibitor, GM6001, H1152 and blebbistatin were from Merck Biosciences (Nottingham, UK). All On-Target Plus Smartpool siRNAs were from Dharmacon, Thermo Fisher Scientific (Fermentas GmbH, St Leon, Germany). Protease and phosphatase inhibitor cocktail were purchased from Roche (Sussex, UK).
3
Isolation of Metacyclic Leishmania
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leishmania major LV39 parasites (MRHO/Sv/59/P strain), mKikume-expressing L. major SWICH (M€ uller et al., 2013), and L. major-mCherry (Ronet et al., 2019) were cultured in M199 medium with 10% fetal bovine serum (PAA Laboratories), 4% HEPES (Amimed) and 2% antibiotics (penicillin, streptomycin, neomycin, Invitrogen) at 26 C. Hygromycin B (PAA Laboratories) at a concentration of 50 mg/mL was added to the medium for the culture of L. major LV39 parasites (MRHO/Sv/59/P strain) expressing the mCherry red fluorescent protein. Metacyclic L. major parasites were isolated using a Ficoll (Sigma) density gradient. Parasites were re-suspended in M199 medium and layered on 10% and 20% Ficoll-medium suspension phases. After centrifugation at 1000 xg for 15 minutes, metacyclic L. major parasites were isolated in the 10% Ficoll phase. The parasites were washed and counted and adjusted to a suitable concentration.
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!