An Immunofluorescence Assay to Characterize and Quantify Mammalian Stress Granules

Abstract

Source: Aulas, A. et al., Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules. J. Vis. Exp. (2017)

This video demonstrates an assay for characterizing and quantifying mammalian stress granules, which consist of mRNAs, RNA-binding proteins (RBPs), and various other proteins.

Protocol

1. Cell Preparation

1. Add autoclaved coverslips to 12 wells of a 24-well plate, as indicated in Figure 1A; this can be done using a sterile Pasteur pipette attached to a vacuum to pick up each coverslip. Gently tap the coverslip on the side of the well to release the suction, allowing the coverslip to fall into the well.
2. Plate 1 x 10⁵ U-2 OS (U2OS) osteosarcoma cells per well at a final volume of 500 µL of medium. Move the plate side to side and up and down several times to ensure that the cells are evenly distributed.
3. Gently press the coverslips down with a clean P1000 tip to ensure that ....

Results

Figure 1: Experimental Diagrams. (A) Seed cells onto coverslips previously placed in a 24-well plate, as indicated. Treat Rows A and B for 60 min with SA or VRB using the concentration in µM as indicated. After 60 min, add CHX (final.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
U-2 OS	ATCC	ATCC®HTB-96™	- commonly written as "U2OS" - culture at 37 °C under 5% CO2 in 10% FBS/DMEM
Dulbecco's Modification of Eagle's Medium	Corning	10-013-CV	- abbreviated DMEM - contains 4.5 g/L glucose, pyruvate, and phenol red - supplemented with 10% FBS, 10 mM HEPES, and penicillin/steptomycin - prewarm prior drug(s) dilution - prewarm prior drug(s) dilution
Fetal Bovine Serum	Sigma	F2442-500ml	- abbreviated FBS - used to supplement media
HEPES (1 M)	Thermo Fisher Scientific	15630-080	- used to supplement media
Penicilline Streptomycine	Sigma	P0781-100ml	- used to supplement media
24 well plate	Costar	3524
Lab tissues	KIMTECH SCIENCE	34120	- commonly called "kimwipes"
Coverglass for Growth Cover Glasses	Fisher Scientific	12-545-82	- autoclaved before used - keep sterile in the tissue culture hood
Sodium (meta)arsenite ≥90%	Sigma	S7400-100G	- commonly called sodium arsenite and abbreviated SA - dissolution in water at 1 M concentration, then dilute to 100 µM as a working stock
Vinorelbine	TSZ CHEM	RS055	- abbreviated VRB - dissolve in water to 10 mM
Puromycin	Sigma	P9620	- used to assess translation level (ribopuromycylation) - used to assess SG connection with active translation - dilute in water to 10 mg/mL
Emetine dihydrochloride hydrate	Sigma	E2375	- used in combination with puro to assess general translation level - used to assess SG connection with active translation - dilute in water to 10 mg/mL
Cycloheximide	Sigma	C4859	- abbreviated CHX - used to assess SG connection with active translation - dilute in water to 10 mg/mL
Phosphate Buffered Saline	Lonza / VWR	95042-486	- abbreviated PBS - wash buffer for immunofluorescence
Paraformaldehyde reagent grade, crystalline	Sigma	P6148-500G	- make a 4% solution in hot PBS in fume hood, stir until dissolved - aliquots can be stored at - 20 °C for several months - hazardous, use ventilation - discard in special waste
Methanol, ACS	BDH / VWR	BDH1135-4LP	- pre-chilled to -20°C before use - discard in special waste
Normal Horse Serum	Thermo Fisher Scientific	31874	- abbreviated NHS - dilute to 5% in PBS - add sodium azide for storage at 4°C - blocking solution for immunofluorescence
Ethanol, Pure, 200 Proof (100%),USP, KOPTEC	Decon Labs / VWR	89125-188	- dilute to 70% with water
Fisherbrand Superfrost Plus Microscope Slides	Fisherbrand	12-550-15
Mouse anti G3BP1 antibody (TT-Y)	Santa Cruz	sc-81940	- store at 4 °C - use at 1/100 dilution
Rabbit anti eIF4G antibody (H-300)	Santa Cruz	sc-11373	- store at 4 °C - 1/250 dilution
Goat anti eIF3η (N-20) antibody	Santa Cruz	sc-16377	- eIF3η is also known as eIF3b - store at 4 °C - 1/250 dilution
Cy2 AffiniPure Donkey Anti-Mouse IgG (H+L)	Jackson Immunoresearch	715-225-150	- reconstitute in water as per manufacturer's instructions then store at 4°C - 1/250 dilution
Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson Immunoresearch	711-165-152	- reconstitute in water as per manufacturer's instructions then store at 4°C - 1/2500 dilution
Cy5 AffiniPure Donkey Anti-Goat IgG (H+L)	Jackson Immunoresearch	705-175-147	- reconstitute in water as per manufacturer's instructions then store at 4°C - 1/250 dilution
Hoechst 33258 solution	Sigma	94403-1ML	- incubate with secondary antibodies-stock solution 0.5 mg/mL in dH20, protect from light- Store at 4 °C
20 x SSC buffer	Thermo fisher Ambion	AM9763	- dilute to 2X SSC using RNase Free water (DEPC treated)- store at room temperature- wash buffer for FISH
Slide mounting media	home made	/	- mix 5 g of "cold-soluble" poly(vinyl alcohol) in 20 ml of PBS- mix by sonication, followed by stirring overnight at RT- Add 5 ml of glycerol and 0.2 ml of 20 % sodium azide, and stir for 16 h at RT- centrifugation at 20,000 g for 20 min, discard large pellet- aliquot viscous liquid, long-term storage at -20°C, 1 week at 4°C
Parafilm "M"	Sigma	P7793-1EA	- usually referred as "parafilm"
Poly(vinyl alcohol)	Sigma	P-8136-250G	- reagent to make vinol
Glycerol	Sigma	G5516-100ML	- reagent to make vinol
Sodium azide	Fisher Scientific	S2002-5G	- preservative agent for blocking solution and vinol - make a 20 % dilution in water