Methods & Materials

Experimental molecular cell Biology

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🧪 Materials & Methods — Full Educational Summary

🐻 5.0 Seasonal Bear Serum Samples

This study investigates seasonal differences in brown bear serum and their effects on C. elegans.

Summer serum (SBS)
- Collected May 2020
- 2-year-old bear, Orsa (Sweden)
- Stored in 5 mL tubes, aliquoted into 2 mL Eppendorf tubes
Winter serum (WBS)
- Collected November 2019
- 2-year-old bear, Borås (Sweden)
- One tube contained a distinct blue layer → treated as a special case

📌 The blue winter serum was characterized biochemically but not used in worm treatments.

🏷️ 5.0.2 Serum Nomenclature

To avoid confusion, each serum condition was clearly named:

Name	Description
PBS control	Untreated control
SBS pure	Undiluted summer serum
SBS 1:10	Summer serum diluted 1:10 in PBS
WBS pure	Undiluted winter serum
WBS 1:10	Winter serum diluted 1:10 in PBS
WBS blue	Winter serum with blue layer (characterization only)

🧠 This naming system is used consistently throughout all experiments.

🔬 Figure 5.1 – Experimental Workflow Overview

A BioRender diagram summarizes the entire pipeline:

Bear serum → NanoDrop & SDS-PAGE
Serum treatments → NGM plates
Multiple C. elegans strains → behavioral, molecular, and imaging assays

This figure acts as the roadmap for the study.

📊 5.1 NanoDrop Analysis of Serum

Goal: Measure protein concentration and spectral properties.

Instrument: NanoDrop 1000
Measurement:
- A280 for protein concentration
- UV-Vis spectra (220–750 nm) for diluted samples
Principle: Beer–Lambert law A = arepsilon cdot l cdot c

🧬 Why 280 nm? Aromatic amino acids (tryptophan, tyrosine) absorb strongly here → standard for protein quantification.

PBS was used as the blank.

🧫 5.2 SDS-PAGE & Coomassie Staining

Goal: Compare protein composition between summer and winter serum.

Sample prep

Dilutions: 1:10, 1:50, 1:100
Centrifugation removes aggregates
Blue winter serum formed a blue pellet + blue supernatant after spin 🟦

Gel preparation

Sample mix: serum + DTT + Laemmli buffer
Heated to denature proteins
Run conditions: 190 V, 400 mA, 1 hour

Visualization

Coomassie Brilliant Blue staining
Destained overnight
Imaged using ChemiDoc MP

🧠 Outcome: visual comparison of serum protein profiles and validation of the unusual blue sample.

🪱 5.3 C. elegans Strains Used

Three strains, each chosen for a specific purpose:

PD4251 🟢
- GFP-labeled mitochondria + nuclei in muscle
- Used for imaging & morphology
NL2099
- RNAi-hypersensitive
- Sterile at high temperature
- Used for thrashing & muscle assays
CB1370
- daf-2 mutant
- Dauer-constitutive at high temperature
- Used for dauer and SDS-resistance assays

🌱 5.3.1 Worm Maintenance

Medium: NGM plates with OP50 E. coli
Regular transfers every 3–4 days
Temperature control:
- PD4251 & CB1370 → 20 °C
- NL2099 → 16 °C

Proper maintenance ensures healthy, synchronized populations.

🥚 5.3.2 Eggs & Serum Spotting

Goal: Ensure worms ingest serum components.

OP50 bacteria UV-killed (no metabolism)
Eggs synchronized and plated in quadruplicates
Each plate spotted with:
- PBS
- SBS (pure / 1:10)
- WBS (pure / 1:10)

🧠 Worms were later analyzed at 3 and 6 days of age.

🐣 5.4 Hatching Assay

Question: Does serum affect egg viability?

100 eggs per condition
Incubated 3 days at 25 °C
Adult progeny counted

Outcome: hatching efficiency under different serum treatments.

🏊 5.5 Thrashing Assay

Purpose: Measure neuromuscular function.

Procedure

Worms placed in M9 buffer
Recorded for 30 seconds
Microscopy + digital video

Analysis

Software: ImageJ (Fiji)
Plugin: wrMTrck
Image processing steps:
- Background subtraction
- MaxEntropy thresholding

🧠 A thrash = full left-right-return body bend.

🎨 5.6 Phalloidin Staining (Actin)

Goal: Visualize muscle actin filaments.

Key steps

Fixation with 4% PFA
Freeze-cracking in liquid nitrogen ❄️
Permeabilization with Triton X-100
Phalloidin (2 µg/mL) staining

Imaging

Confocal microscopy (Olympus IX83)
RFP channel (560 nm laser)

🧬 Reveals muscle integrity and structure.

🧬 5.7 Western Blot (β-Actin)

Purpose: Quantify actin levels in worms.

Sample: 30 worms per condition
SDS-PAGE → nitrocellulose transfer
Detection:
- Primary: anti-β-actin
- Secondary: HRP-conjugated antibody
Signal: chemiluminescence (ECL)

🔁 Membrane stripped and reprobed with α-tubulin as loading control.

🟢 5.8 GFP Visualization

Used PD4251 strain

Worms mounted on agarose
Immobilized with levamisole
Confocal imaging:
- 488 nm laser
- Mitochondria + nuclei in muscle cells

Provides direct visualization of mitochondrial structure.

📐 5.8.1 Quantitative Mitochondrial Morphology

Two metrics used:

1️⃣ Eccentricity

Measures elongation
0 = round, 1 = perfectly tubular

2️⃣ Skeletonized Branch Density

Uses AnalyzeSkeleton plugin
Quantifies network complexity

🧠 Together: shape + connectivity = robust morphology analysis.

👀 5.8.2 Qualitative Morphology Scoring

Blind scoring into three categories:

Fragmented 🔴
Intermediate 🟡
Tubular 🟢

Whole anterior body analyzed (not just pharynx region).

💤 5.9 Dauer Experiment

Strain: CB1370 (daf-2)

Eggs exposed to serum conditions
Incubated 48 h
Dauer verification via 1% SDS resistance
- Survivors = dauers
- Movement checked manually

🧠 Dauer formation reflects metabolic and stress responses.

✅ Big Picture Takeaway

This methods section combines:

🧪 Biochemistry (NanoDrop, SDS-PAGE)
🪱 Whole-organism assays (thrashing, dauer)
🧬 Molecular biology (Western blot)
🧠 Advanced imaging & quantitative morphology

All methods are tightly integrated to test how seasonal bear serum influences muscle function, mitochondrial structure, and stress resilience in C. elegans.

Quiz

Score: 0/30 (0%)

Q0. What was the primary purpose of using seasonal brown bear serum in this study?

To compare bacterial growth rates

To investigate seasonal effects on muscle, mitochondrial, and stress-related phenotypes in C. elegans

To validate NanoDrop protein quantification

To induce dauer formation directly through serum toxicity

Q1. Which serum sample displayed a distinct blue layer after centrifugation?

SBS pure

SBS 1:10

WBS pure

WBS blue

Q2. Why was the WBS blue serum not used as a treatment group for C. elegans assays?

It had too low protein concentration

It was contaminated with bacteria

Its unusual composition could confound biological interpretation

It could not be diluted in PBS

Q3. Which wavelength was used for protein concentration measurements in the NanoDrop analysis?

260 nm

280 nm

320 nm

750 nm

Q4. Which law underlies the calculation of protein concentration from absorbance values?

Michaelis–Menten equation

Beer–Lambert law

Hooke’s law

Ohm’s law

Q5. What was the purpose of including DTT in the SDS-PAGE sample preparation?

To stain proteins

To reduce disulfide bonds

To increase gel polymerization

To fix proteins in place

Q6. Which staining method was used to visualize proteins after SDS-PAGE?

Silver staining

Western blotting

Coomassie Brilliant Blue

Fluorescent antibody staining

Q7. Which C. elegans strain was used specifically for mitochondrial GFP visualization in muscle cells?

NL2099

CB1370

PD4251

Q8. Why were OP50 bacteria UV-killed before serum exposure on NGM plates?

To improve worm feeding behavior

To prevent bacterial metabolism of serum components

To increase bacterial fluorescence

To sterilize the agar

Q9. What biological outcome was measured in the hatching assay?

Thrashing frequency

Mitochondrial morphology

Egg-to-adult survival rate

Protein expression levels

Q10. What does one full thrash represent in the thrashing assay analysis?

One head movement

One left-right-return body bend

One second of movement

One contraction of body wall muscle

Q11. Why was background subtraction applied during thrashing video analysis?

To increase video resolution

To remove constant, non-moving elements

To enhance fluorescence intensity

To normalize worm size

Q12. What is the primary target of phalloidin staining in this study?

Microtubules

Intermediate filaments

F-actin

Mitochondrial membranes

Q13. Why was freeze-cracking used during worm fixation for phalloidin staining?

To enhance fluorescence

To permeabilize the cuticle

To preserve mitochondrial morphology

To remove lipids

Q14. What was the main purpose of using α-tubulin in the Western blot?

As a positive control for serum proteins

As a mitochondrial marker

As a loading control

As a secondary antibody

Q15. True or False: The NL2099 strain becomes sterile at high temperatures due to increased RNAi sensitivity.

True

False

Q16. True or False: PBS was used as both a dilution buffer and an untreated control.

True

False

Q17. True or False: NanoDrop UV-Vis spectra were collected only at 280 nm.

True

False

Q18. True or False: The SDS-PAGE gel was run at constant voltage and current for one hour.

True

False

Q19. True or False: CB1370 worms form dauers constitutively at elevated temperatures.

True

False

Q20. True or False: Thrashing assays were performed on both 3-day-old and 6-day-old worms.

True

False

Q21. True or False: Phalloidin staining was visualized using GFP excitation settings.

True

False

Q22. True or False: Western blot membranes were blocked with skim milk to reduce non-specific binding.

True

False

Q23. True or False: GFP imaging was performed using a spinning disk confocal microscope.

True

False

Q24. True or False: An eccentricity value of 0 represents a perfectly tubular mitochondrion.

True

False

Q25. True or False: Skeletonized branch density reflects mitochondrial network connectivity.

True

False

Q26. True or False: Qualitative mitochondrial morphology scoring was performed using a blinded approach.

True

False

Q27. True or False: Only the region between the two pharyngeal bulbs was analyzed for mitochondrial morphology.

True

False

Q28. True or False: Dauer formation was confirmed using SDS resistance.

True

False

Q29. True or False: Movement was used to distinguish live from dead worms in the dauer assay.

True

False