In this study, a reference fungal strain C. albicans obtained from American Type Culture Collection (ATCC 90028) was used. For maintenance, thawed suspension from glycerol stock (30%) stored at ultra-low deep-freezing condition (−80°C) was streaked on yeast extract peptone dextrose (YEPD) agar plates (1.8%) and incubated at 37°C for 24 h and stored at 4°C for further use. Routine culturing for in vitro assays was performed with YEPD broth. In vitro assays allied with biofilm formation, and hyphal development was carried out with Spider medium comprising 1% of mannitol, 0.2% of dipotassium hydrogen phosphate, and 1% of nutrient broth. To investigate the effect of piperine on the preformed hyphae of C. albicans, RPMI medium (HiMedia, India) was used.
Rpmi medium
Manufactured by HiMedia
Sourced in India
RPMI medium is a cell culture medium commonly used for the in vitro cultivation of a variety of cell types, including human and animal cells. It provides the essential nutrients and growth factors necessary to support the proliferation and maintenance of these cells in a controlled laboratory environment.
Automatically generated - may contain errors
4 protocols using rpmi medium
1
Antifungal Effects of Piperine on C. albicans
2
Isolating human PBMCs from blood
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The blood sample (5 ml) was collected from a healthy human volunteer in a tube containing EDTA. Equal volume of lymphocyte separation medium (Histopaque-HiMedia, India) was taken in a fresh tube upon which the freshly collected blood was layered carefully and subjected to centrifugation at 2200 rpm for 30 minutes at 20°C. After removing the upper layer, the buffy coat mononuclear layer was carefully transferred to a fresh tube and washed using RPMI-1640 (Roswell Park Memorial Institute) medium at 2200 rpm for 15 min at 20°C. The pellet was resuspended in the complete medium [RPMI medium (HiMedia, India), 10% fetal bovine serum (HiMedia, India) and 1% Anti-Anti (Antibiotic-Antimycotic) solution (Invitrogen, United States)] and the cell viability was assessed through trypan blue exclusion assay. PBMCs were adjusted to 1 × 106 cells ml–1 in complete medium for further experiments (Syad and Kasi, 2014 (link)).
3
Cytotoxicity Evaluation of P. rosea on Melanoma and Lymphocytes
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SK-MEL 28, human melanoma cell lines were procured from National Centre for Cell Science (NCCS), Pune, India. Cells were cultured in DMEM medium supplemented with 10% heat inactivated FBS, 1% glutamine, and 1% penicillin. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Human lymphocytes culture was used to evaluate the cytotoxicity of P. rosea. Lymphocytes were isolated using HiSep (HiMedia, India) as per the manufacturer's instruction. Briefly, HiSep media was added to the blood in the ratio 1:3 (media: blood), and centrifuged at 160 g for 20 min. The lymphocytes were then transferred into fresh tube and washed with sterile PBS by centrifuging at 140 g for 15 min. The pellet were cultured in RPMI medium (HiMedia) supplemented with 10% heat inactivated bovine serum, antibiotics (streptomycin, penicillin), NaHCO3. Phytoheamagglutinins were used as a mitogen for the proliferation of lymphocytes. The culture was filtered by using sterilized 0.2-μm pore size cellulose acetate membrane filter (Sartorius). Isolated human peripheral lymphocytes were incubated for 72 h at 37°C.
Human lymphocytes culture was used to evaluate the cytotoxicity of P. rosea. Lymphocytes were isolated using HiSep (HiMedia, India) as per the manufacturer's instruction. Briefly, HiSep media was added to the blood in the ratio 1:3 (media: blood), and centrifuged at 160 g for 20 min. The lymphocytes were then transferred into fresh tube and washed with sterile PBS by centrifuging at 140 g for 15 min. The pellet were cultured in RPMI medium (HiMedia) supplemented with 10% heat inactivated bovine serum, antibiotics (streptomycin, penicillin), NaHCO3. Phytoheamagglutinins were used as a mitogen for the proliferation of lymphocytes. The culture was filtered by using sterilized 0.2-μm pore size cellulose acetate membrane filter (Sartorius). Isolated human peripheral lymphocytes were incubated for 72 h at 37°C.
4
Cell Line Cultivation and Maintenance
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HepG2, HeLa, MCF7, Jurkat and K562 cell lines were procured from National Centre for Cell Sciences, Pune, India. HepG2, HeLa and MCF 7 cells were maintained in MEM medium (HIMEDIA, India), Jurkat and K562 in RPMI medium (HIMEDIA, India) supplemented with 10% Fetal Bovine Serum (HIMEDIA, India). The cells were incubated in a humidified incubator at 37°C with 95% air and 5% CO2 (Thermo Scientific, USA).
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