Korea-CX

Experiments

Experiments Day 1: Cell Culture

<Part 1. Basic Theory of Cell Culture>

Experimental Principle

• Definition: Cell culture is the process of growing live cells in a controlled, artificial environment outside the body.
• Purpose: To study cell behavior, drug responses, and genetic changes.
• Cell Types: Immortalized cell lines such as NIH3T3 are commonly used.
• Immortalized Cells: These are cells engineered to divide indefinitely by overcoming senescence, often using viral genes, oncogenes, or telomerase.

Essential Components

1. Media: Provides nutrients
2. FBS: Serum supplying growth factors (10% v/v).
3. Penicillin-Streptomycin: Prevents bacterial contamination (1% v/v).
4. Trypsin-EDTA: Enzyme for detaching adherent cells.
5. PBS: For washing cells.

<Part 2. Cell Culture Procedures>

Materials Needed

• Frozen NIH3T3 cells in cryovials
• DMEM + 10% FBS + 1% PenStrep
• Trypsin-EDTA
• PBS
• Hemocytometer & Trypan Blue
• 6/24/96 well plates, T-flasks

Procedure

1. 1. Cell Thawing
   • Thaw cryovial in 37 °C water bath (2 min).
   • Dilute into warm media, centrifuge (1350 rpm, 3 min).
   • Resuspend in fresh media, transfer to T-flask, incubate at 37 °C, 5% CO₂.
2. 2. Subculturing (Passaging)
   • Remove old media, wash with PBS.
   • Add trypsin, incubate 1–3 min until cells detach.
   • Neutralize with media, collect cells, centrifuge, resuspend, and split.
3. 3. Media Change
   • Replace old media with fresh pre-warmed DMEM.
4. 4. Cell Seeding
   • Count cells using hemocytometer + Trypan Blue.
   • Calculate volume for target density.
   • Seed into plates or chips for downstream assays.
5. 5. Cell Freezing (Stocking)
   • Harvest cells, centrifuge.
   • Resuspend in freezing media (90% FBS + 10% DMSO).
   • Aliquot into cryovials, freeze at -80 °C, transfer to LN₂ for long-term storage.

<Part 3. Practice Skills>

1. Pipetting Practice
   • Use micropipettes (P10, P20, P200, P1000).
   • Accuracy check with water-weighing (100 µL = 0.100 g, ±0.005 g).

   

2. Cell Counting with Trypan Blue
   • Mix cells and Trypan Blue 1:1.

   

   • Load into hemocytometer (10 µL).

   

   • Count 4 squares, calculate concentration and viability.

   

3. Seeding in 96-well Plate
   • Determine seeding density (e.g., 2 × 10⁴ cells/well).
   • Calculate suspension volume, prepare master mix.
   • Dispense evenly, avoid bubbles, incubate at 37 °C, 5% CO₂.

   

   

   

Experiments Day 2: Plasmid Transfection

<Part 1. Basic Theory of Plasmid Transfection>

Experimental Principle

• Definition: Plasmid transfection is the process of introducing foreign DNA into eukaryotic cells using non-viral methods (e.g., lipofection or electroporation).
• Purpose: To induce gene expression of wild-type or mutant NRAS (WT vs G12D) and visualize successful delivery via mCherry fluorescence.
• Application: This enables downstream analysis of cell viability, migration, and drug sensitivity in genetically modified cells.
• Plasmid Preparation: Plasmids encoding NRAS^WT and NRAS^G12D with an mCherry reporter were synthesized and directly introduced into NIH/3T3 cells via Lipofectamine-mediated transfection. Stable pools were generated by puromycin selection.

Key Concepts

• NRAS Mutation (G12D): A point mutation leading to constitutive activation of NRAS signaling.
• Experimental Groups:
  • Control : Untransfected NIH3T3 cells
  • Wild-type group : NIH3T3 cells transfected with NRAS-WT plasmid (with mCherry reporter)
  • Mutant group : NIH3T3 transfected with NRAS-G12D plasmid (with mCherry reporter)
• Fluorescence Check : Successful transfection is verified by mCherry fluorescence.
• Transfection Methods:
  • Lipofection: DNA-lipid complexes merge with cell membranes.

<Part 2. Transfection Procedures>

Materials Needed

• NIH3T3 cells (60–80% confluency)
• Plasmids: NRAS-WT, NRAS-G12D (with mCherry reporter)
• Lipofectamine™ 3000 + Opti-MEM (for lipofection)
• DMEM + 10% FBS + 1% PenStrep
• PBS, Trypsin-EDTA, T-flasks, 6-well plates
• Sterile tubes, pipettes, tips

Procedure (Lipofection Method)

1. 1. Cell Preparation
   • Seed NIH3T3 cells in 6-well plates (2–3 × 10⁵ cells/well).
   • Ensure ~70% confluency on the day of transfection.
2. 2. DNA–Lipid Complex Formation
   • Tube A: Dilute 2.5 µg plasmid DNA + 5 µL P3000 reagent in 125 µL Opti-MEM.
   • Tube B: Dilute 7.5 µL Lipofectamine 3000 in 125 µL Opti-MEM.
   • Combine Tube A and B, mix gently, incubate 10–15 min at room temperature.
3. 3. Transfection
   • Add mixture dropwise to each well containing 2 mL fresh medium.
   • Gently swirl plate to distribute complexes evenly.
4. 4. Incubation
   • Incubate 6 h at 37 °C, 5% CO₂.
   • Replace with fresh complete media.
   • Maintain for 24–48 h before downstream assays.

<Part 3. Practice Skills>

1. 1. Plasmid Handling
   • Always keep plasmids on ice during setup.
   • Measure DNA purity with NanoDrop (A260/A280 ~1.8–2.0).

   

2. 2. Transfection Controls
   • Replace media to reduce cytotoxicity.
   • Monitor cells daily for morphology changes.
3. 3. Post-Transfection Care
   • Replace media to reduce cytotoxicity.
   • Monitor cells daily for morphology changes.

Experiments Day 3: Gene Expression Analysis

<Part 1. Basic Theory of Gene Expression Analysis>

Experimental Principle

• Definition: Gene expression analysis measures whether transfected plasmids are expressed at RNA and protein levels.
• Purpose: To confirm successful delivery of NRAS-WT and NRAS-G12D plasmids in NIH3T3 cells.
• Techniques:
  • Fluorescence Microscopy for mCherry reporter expression.
  • RT-PCR to detect NRAS transcripts.
  • Gel Electrophoresis to visualize amplified products.

Key Concepts

• mCherry Reporter: Red fluorescence indicates successful plasmid uptake.
• RNA → cDNA → PCR: Stepwise workflow for validating transgene expression.
• Internal Control (GAPDH): Ensures accuracy by normalizing expression levels.

<Part 2. Experimental Procedures>

Materials Needed

• NIH3T3 cells transfected with plasmids (Exp. 2)
• Fluorescence microscope with mCherry/Texas Red filter
• Invitrogen PureLink RNA Mini Kit
• Bioneer RocketScript RT Premix (cDNA synthesis)
• PCR premix, GAPDH/NRAS primers
• NanoDrop spectrophotometer
• Agarose gel, TAE buffer, DNA ladder, Gel doc system

A. Fluorescence Microscopy

1. Wash cells with PBS, add fresh media.
2. Focus under brightfield, switch to mCherry filter.
3. Capture images at multiple fields; record % positive cells.

B. RNA Extraction & cDNA Synthesis

1. Harvest 1 × 10⁶ cells, lyse with buffer + β-mercaptoethanol.
2. Bind RNA to column, wash, elute in RNase-free water (50 µL).
3. Quantify RNA with NanoDrop (260/280 ~1.8–2.1).
4. Use 500 ng RNA for cDNA synthesis (50 °C, 30 min → 95 °C, 5 min).

C. PCR Amplification

• Reaction (20 µL): cDNA template, GAPDH primer (internal control), NRAS-WT or NRAS-G12D primer, premix, water.
• PCR program:
  • Initial denaturation : 95 °C 1 min
  • Cycling : 95 °C 15 s, 55 °C 15 s, 72 °C 30 s) × 35 cycles
  • Final extension : 72 °C 7 min.
  • Hold: 4 °C

  • 

D. Gel Electrophoresis

1. 1. Prepare 2% agarose gel with nucleic acid stain.
2. 2. Load 15 µL PCR product + 4 µL ladder.
3. 3. Run at 130 V for 30 min.
4. 4. Visualize under gel doc; confirm expected band sizes.

<Part 3. Practice Skills>

Materials Needed

• NIH3T3 cells transfected with plasmids (Exp. 2)
• Fluorescence microscope with mCherry/Texas Red filter
• Invitrogen PureLink RNA Mini Kit
• Bioneer RocketScript RT Premix (cDNA synthesis)
• PCR premix, GAPDH/NRAS primers
• NanoDrop spectrophotometer
• Agarose gel, TAE buffer, DNA ladder, Gel doc system

• Microscopy
  • Avoid photobleaching by minimizing light exposure.
  • Use the same exposure time for all groups.
• RNA Work
  • Always use RNase-free consumables.
  • Keep samples on ice during prep.
• PCR & Gel
  • Include no-template control (NTC) to check contamination.
  • Compare band intensity between WT and G12D for expression lev

Experiments Day 4: CCK-8 Drug Response Assay with MEK inhibitor U0126

<Part 1. Basic Theory of CCK-8 Assay>

Experimental Principle

• Definition: The CCK-8 assay is a colorimetric method to determine cell viability based on metabolic activity.
• Purpose: To evaluate the cytotoxic effect of the MEK inhibitor U0126 on cultured cells by measuring dose-dependent viability.
• Mechanism: The CCK-8 reagent contains WST-8, a tetrazolium salt reduced by dehydrogenases in metabolically active cells to form a soluble orange formazan dye.

The intensity of color (absorbance at 450 nm) is directly proportional to the number of viable cells.

About U0126

• U0126 is a selective inhibitor of MEK1 and MEK2, components of the MAPK/ERK signaling pathway. It is widely used in research to block ERK phosphorylation and study cell proliferation, survival, and differentiation.
• U0126 Concentration Calculation
  U0126 MW = 380.4 g/mol

Basic formula:
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀) = 𝑚𝑎𝑠𝑠 (𝑔) / 𝑀𝑊 (𝑔/𝑚𝑜𝑙) × 𝑣𝑜𝑙𝑢𝑚𝑒 (𝐿)

Example:
To prepare a 10 mM stock solution in DMSO:
  

      
Desired final volume: 1 mL
        
Target concentration: 10 mM
        
Required mass of U0126:

              10𝑚𝑀×380.4𝑔/𝑚𝑜𝑙×0.001𝐿 = 3.804𝑚𝑔

<Part 2. Experimental Procedures>

Materials Needed

• NIH3T3 cells
• U0126 stock solution (10 mM in DMSO)
• CCK-8 solution (Cell Counting Kit-8)
• 96-well plate
• DMEM with 10% FBS + 1% Pen/Strep
• Plate reader (450 nm)
• Sterile tips, pipettes, conical tubes

Procedure

1. 1. Cell Seeding
   • Prepare cell suspension (1 × 10⁴ cells/100 µL/well).
   • Seed into a 96-well plate (100 µL/well).
   • Incubate 24 h at 37 °C, 5% CO₂ before drug treatment.
2. 2. Drug Dilution & Treatment
   • Prepare serial dilutions of U0126 in medium from 10 mM stock.
   • Example dilution series (final concentrations, µM): 40, 20, 10, 5, 2.5
   • Add 100 µL of each dilution to triplicate wells.
   • Include control wells with vehicles (DMSO only).
   • Incubate cells for 24 h.

   

   

3. CCK-8 Addition
   • Add 20 µL CCK-8 reagent to each well (final volume 120 µL).
   • Gently tap the plate to mix.
   • Incubate 2 h at 37 °C (protect from light).

   

4. Absorbance Measurement
   • Wipe bottom of plate with Kimwipe + ethanol.
   • Measure absorbance at 450 nm using a microplate reader.
   • Record triplicate readings for each condition.

   

<Part 3. Practice Skills>

1. Serial Dilution & Pipetting
   • Practice preparing serial dilutions using Trypan Blue or dye before working with U0126.
   • Always mix thoroughly before transferring to the next tube.
2. Assay Controls
   • Include blank wells (media + CCK-8, no cells) for background correction.
   • Use vehicle-only wells (DMSO without drug) as 100% viability control.
3. Data Reliability
   • Run samples in triplicates for each condition.
   • Avoid bubbles in wells, as they interfere with absorbance readings.
   • Normalize data against control wells.

   𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝐴𝑏𝑠.𝑠𝑎𝑚𝑝𝑙𝑒 / 𝐴𝑏𝑠.𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100

Experiments Day 5: Lab-on-a-Chip (LOC) Fabrication

<Part 1. Basic Theory of Lab-on-a-Chip (LOC)>

Experimental Principle

• Definition: A Lab-on-a-Chip (LOC) is a microfluidic platform integrating laboratory functions into a millimeter–centimeter-sized device.
• Purpose: To fabricate a PDMS-based LOC that allows cell seeding, migration assays, and drug testing in controlled microenvironments.
• Advantages:
  • Minimal reagent/sample consumption
  • Real-time monitoring under microscopy
  • Mimics physiological structures (tissue, vasculature, tumor)
  • Enables flow control and chemical gradient formation

Why PDMS?

• Transparent → ideal for microscopy
• Biocompatible and gas permeable
• Easy to mold (soft lithography)
• Hydrophobic (temporarily hydrophilic after plasma treatment for bonding)

Chip Design Overview

• Layer 1 (Reservoir): Media exchange & drug delivery (5 × 8 mm circles, 4 mm thick).
• Layer 2 (Chamber Top): Cell seeding chamber (6 mm central well, aligned with porous membrane).
• Layer 3 (Chamber Bottom + Channels): Collection chamber with microchannels, mimicking vasculature.
• Porous Membrane: Inserted between layers to simulate biological barriers.
• Layer 4 (Glass Slide): Bottom support, allowing imaging.

<Part 2. Experimental Procedures>

Materials Needed

• PDMS base + curing agent (10:1 ratio)
• Silicon molds for each layer
• Biopsy punches (1, 2, 6, 8 mm)
• Plasma surface treatment system
• Glass slides
• Transwell membrane insert
• 70% EtOH, PBS, DMEM

1. 1. PDMS Preparation
   • Mix PDMS base:curing agent (10:1).
   • Pour into molds; de-gas under vacuum.
   • Cure at 60 °C for 3–4 h (overnight preferred).

   

   

2. 2. Layer Cutting & Punching
   • Remove cured PDMS layers.
   • Punch reservoirs, chambers, inlets/outlets (sizes: 1–8 mm).

   

3. 3. Plasma Bonding
   • Cut membrane from transwell insert.
   • Plasma-treat each PDMS layer + glass slide.
   • Align and bond in order: Layer 3 → Layer 2 → Membrane → Layer 1 → Glass slide.

   

   

4. 4. Chip Washing & Conditioning
   • Leave overnight for hydrophobic recovery.
   • Wash sequentially: 70% EtOH → PBS → DMEM.
   • Check flow uniformity and absence of leakage.

   

<Part 3. Practice Skills>

• Soft Lithography Handling
  • Avoid bubbles during PDMS pouring/de-gassing.
  • Use consistent curing times for reproducible thickness.
• Membrane Alignment
  • Handle membrane with forceps, avoid folding/tearing.
  • Ensure central alignment with chambers.
• Plasma Surface Treatment
  • Settings used: O₂ gas 45 sccm, RF power 90 W, 1 min.
  • Immediately bond after treatment (hydrophilicity is temporary).

Experiments Day 6: 3D Bone Marrow Microenvironment & Migration Assay

<Part 1. Basic Theory>

Experimental Principle

• Definition: Construction of a 3D bone marrow-like microenvironment in LOC to study cancer cell migration.
• Purpose: To mimic ECM and stromal interactions using Matrigel and track cancer cell behavior under realistic conditions.

Background

• Bone Marrow Niche: Provides signals (stromal cells, ECM proteins, cytokines) that regulate hematopoietic and malignant cell behavior.
• Matrigel: Basement membrane extract rich in laminin/collagen IV, forming ECM scaffolds that support 3D growth.
• LOC + Matrigel: Combines fluidic control with ECM simulation → closer to in vivo.

Note: NIH3T3 fibroblasts were used as a model system instead of human multiple myeloma cells, in compliance with iGEM safety guidelines.

<Part 2. Experimental Procedures>

Materials Needed

• LOC device (from Exp. 5)
• Matrigel (thawed on ice, 4 mg/mL final conc.)
• NIH3T3
• DMEM + 10% FBS + 1% Pen/Strep
• PBS, 70% EtOH
• CO₂ incubator
• Ice bucket, micropipettes with pre-cooled tips, sterile dishes, timer

1. 1. Chip Preparation
   • Wipe LOC surface with 70% EtOH.
   • Wash channels: EtOH → PBS → medium (repeat twice).
   • Fill channels with medium; condition for 30 min.
2. 2. Matrigel Injection
   • Thaw Matrigel on ice.
   • Load with pre-cooled tips.
   • Slowly inject into the ECM chamber (avoid bubbles).
   • Incubate 37 °C, 30–45 min until polymerized.
3. 3. Cell Preparation & Seeding
   • Harvest NIH3T3 cells; resuspend at 1 × 10⁵ cells/mL.
   • Seed 100 µL of suspension above the Matrigel layer.
   • Fill reservoirs with medium.

   

4. 4. Incubation & Observation
   • Incubate LOC in 37 °C, 5% CO₂ for 24–72 h.
   • Monitor cell morphology and migration under fluorescence or brightfield microscopy.
   • To mimic dynamic body-fluid flow, the LOC was placed on a low-speed rocking shaker inside the incubator. This gentle shaking maintained continuous medium circulation and shear stress similar to physiological conditions.

   

<Part 3. Practice Skills>

• 1. Matrigel Handling
  • Always keep on ice; pre-cool tips/tubes.
  • Avoid repeated freeze–thaw; aliquot before use.
• 2. Injection Technique
  • Avoid bubble formation during Matrigel filling.
  • Ensure uniform ECM distribution for reproducible migration results.
• 3. Imaging & Analysis
  • Capture time-lapse images for migration tracking.
  • Use fluorescence (mCherry) to distinguish transfected cells.