Protocol - Pharmaceutical Cosmetic Science Based Botanical Drug Research Lab.

1. Cell culture
1.1.Cell culture medium
a. DMEM – Dulbecco’s Modified Eagle Medium – NHDFs, HaCaTs, Raw 264.7 cells, Basophil
- Full medium (for subculture and seeding): DMEM containing 10% FBS (Fetal Bovine Serum), 1%
AB (Antibody)

- DMEM: high glucose liquid media supports cell growth in research. DMEM media contain
increased concentrations of vitamins, minerals, and amino acids compared to traditional basal
media. DMEM medium composition also includes ferric nitrate, making this cell culture medium
an option for certain cell lines requiring iron for cell growth. DMEM media preparations are
available in high or low glucose formulations, as well as with or without L-glutamine,
magnesium, and sodium pyruvate. DMEM formulation options may also include phenol red as a
pH indicator. All available DMEM media products contain a sodium bicarbonate buffer with or
without HEPES to maintain optimal culture pH.
- FBS: High in growth factors to support growth and division of cells
- Antibiotics: Penicillin acts by inhibiting bacterial cell-wall synthesis. Streptomycin inhibits
prokaryote protein synthesis by preventing the transition from initiation complex to chain-
elongating ribosome and causes miscoding. Amphotericin B interferes with fungal membrane
permeability by forming channels in the membranes and causing small molecules to leak out.
(Gibco® Antibiotic-Antimycotic 는 세균과 진균 오염 방지를 위해 사용합니다. 그람
양성균과 그람 음성균에 각각 효과적인 활성을 보이는 penicillin 와 streptomycin 항생제
복합체를 세포 배양 시 세균 오염을 방지하기 위해 사용할 수 있습니다. Fungizone®
(amphotericin B)은 다세포 진균과 효모를 억제하여 세포 배양 시 진균 오염 방지에
사용합니다.)
- AB medium (for treatment): DMEM containing 1% AB (Antibody)
- Prepare bottles and filter by sterilizing in UV culture hood (because 1 filter can filter 2 L of
medium, each time you make AB medium, please prepare to make 2L for maximum)

- For 1 L bottle of AB medium: 1 pack powder medium + 3.7 g sodium bicarbonate (NaHCO3) + 1 L
3D water -> shake well until all powder are dissolved
- Sonicate bottle in 15 – 20 mins
- Bring bottles into culture hood and use filter to transfer fresh medium into new bottles

- After filtration, add 12 drops of HCl into 1 L of medium to reach pH 7.0 – 7.3 (Use glass tips in
metal box inside the hood, be careful when working with acid)
- Finally, add 10 mL AB into medium
1.2. Cell lines
a. NHDFs
- NHDFs (Normal Human Dermal Fibroblasts) were obtained from a skin biopsy of a healthy young
male donor at 20 and 40 age (MCTT Core, Inc., Seoul, Korea). NHDF were cultured in 100-mm
dishes in full DMEM at 37°C in a humidified atmosphere containing 5% CO2. NHDF cells were
used between passages 5 and 10.
- Seeding concentration for 35 mm disc 1x10 5 cells/mL
b. HaCaTs
- HaCaTs (Human aneuploid immortal keratinocytes) were purchased from the Korea cell line
bank (Seoul, Korea). HaCaT cells were grown in full DMEM at 37°C in a humidified atmosphere
containing 5% CO2.
- Seeding concentration for 35 mm disc: 2 – 3 x 10 5 cells/mL

c. Raw 264.7 cells

RAW 267.4 cells (1×105/ml cells/well; 2 ml medium added per well in 24-well plates) were cultured
in DMEM containing 10% FBS and 1% penicillin-streptomycin for 3 days for 3 days when confluence
was reached in a water-saturated atmosphere containing 5% CO2 and 95% air at 37°C.

d.
e.  
f.