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Koliwad Lab

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by

Emily Dong

on 8 August 2014

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Transcript of Koliwad Lab

Summer at UCSF
Overview
AIM 1: Activation of Hypothalamic Microglia by Fatty Acids
Saturated FA
Palmitic Acid (100, 300, 500 μM)
Monounsaturated FA
Oleic Acid (100, 300, 500 μM)
BSA
LPS (+)
Mixed standardized sample amounts with sample buffer solution
Make, load, and run polyacrilamide gels
Transfer gel onto membranes
Block non-specific sites
Primary antibodies = P65, PIKK B
Secondary antibody (from rabbit)
Substrate
Chemiluminescent detection
1. Microglia Isolation/RAW264.7 Prep
2. FA Treatment
3. Western Blot
RAW 264.7
Quickly thaw frozen cells
Slowly add warmed media and incubate
Split cells when 80% confluent
Scrape, count, and plate
Microglia
Pups (P2-P4)
Dissection:
Isolate (3) brains in petri dish with HBSS
Isolate cerebral hemispheres by removing cerebellum and meniges
Cell Prep:
Homogenize brain with media + growth factor
Change media (day 3), harvest (day 7-10)
Harvest:
Shake for 1hr. at 200rpm at 37 degrees Celsius (separate astrocytes and microglia)
Take media, spin down to cells, count, plate
Related...
Cultured Fibroblast Cells
Bone Marrow Dissection
Related...
Thin Layer Chromatography
Scintillation Counter- radioactivity
Phosphor Imager- reads TLC
Protein Quantification:
Mix FA treated samples with BCA Protein Assay (duplicates)
Compare with standards using plate reader to standardize protein amount
Nanodrop
shows differences in DNA/RNA/protein concentrations by measuring wavelength
Related...
Differentiate
P17
Monocytes
Human Adipose Tissue
Cafeteria
Pics of KO mice on HFD
* Complex FA to BSA (4:1 molar ratio) for microglial cells- more sensitive to BSA
* Complex FA to BSA (2:1 molar ratio)
Incubate cells with DMEM w/o FBS for 1 hr before FA treatment
Add FA solution (BSA, FA, DMEM w/o FBS) into corresponding wells
Incubate for 30 min
1. Microglia/RAW264.7 Isolation
2. FA Treatment
3. Western Blot
Sample Prep:
RIPA lysis buffer
Scrape, centrifuge, and collect supernatant
*Measure total protein to confirm equal amounts in each sample
Related...
Other ways to detect signaling:
qPCR- mRNA
ELISA- secreted proteins
Microglia
RAW264.7
(300 μM)
(100, 300, 500 μM)
Full transcript