Isolation and Digestion of Neutrophil Proteins

Protocol

1. Neutrophil Cell Collection and Lysis 

1. Collect neutrophil-differentiated HL60 cells in a 15 mL tube by centrifuging samples at 400 x g for 5 mins. 
   NOTE: Neutrophils should be at a confluency of 3.5 x 10⁵ cells/mL after differentiation.  Harvest approximately five million cells for total proteome extraction. 
2. Discard the supernatant, and add 15 mL of phosphate-buffered solution (PBS) into the tube.
3. Centrifuge samples at 400 x g for 5 min.
4. Repeat steps 1.2 to 1.3. 
   NOTE: Cell samples can be flash frozen with ....

Results

Figure 1. Workflow of HL60 cells differentiated into neutrophils for mass spectrometry-based proteomics profiling. Undifferentiated and differentiated (via DMSO chemical stimulation) HL60 cell lines were cultured, collected, and processed for total proteome extraction. Proteins were digested with Trypsin/LysC, and peptides were purified using C18 STAGE-tips, followed by detection by LC-MS/MS. Max.......

Materials

Name	Company	Catalog Number	Comments
15 ml Falcon Tube	VWR	470225-000
25 mL Serological	Fisher Scientific	14955235
Ammonium Bicarbonate (ABC)	ThermoFisher Scientific	A643-500
Acetone	Sigma Aldrich	34850-1L
BSA assay kit	VWR	CA62408-940
Centrifuge 5810 R	Sigma Aldrich	EP022628188
Dithiothreitol (DTT)	ThermoFisher Scientific	R0861	Prepare 1M stock of DTT aliquots and store at  -20°C
Fetal Bovine Serum (FBS)	ThermoFisher Scientific	12483020	Thaw out, prepare aliquots and stored at -20°C
Heating block
HEPES	Sigma Aldrich	H3375	Prepare 8M Urea/40m HEPES aliquots, store at -20 °C
Iodoacetamide  (IAA)	Fisher Scientific	AAA1471514	Prepare 0.55M stock of IAA aliquots and store at  -20°C. Avoid light exposure
L-glutamine	Fisher Scientific	25030081	Thaw out, prepare aliquots and stored at -20°C
Liquid nitrogen
LoBind Microcentrifuge tubes	Eppendorf	13-698-794
Penicillin Streptomycin, Amphotericin	Sigma Aldrich	A5955	Thaw out, prepare aliquots and stored at -20°C
Neutrophil-differentiated HL60 cells
Phosphate-buffered solution (PBS)	Bioshop	PBS408	Store at room temperature
Probe sonicator	Fisher Scientific	100-132-894
Protease inhibitor cocktail (PIC) tablet	Sigma Aldrich	5892791001	Store at 4°C
RPMI 1640	ThermoFisher Scientific	11875093	Store at 4°C
Sodium Dodecyl Sulphate (SDS)	ThermoFisher Scientific	J18220.A1	prepare 2% SDS solution
SpeedVac Centrifuge	Eppendorf	07-748-15
T75 Flasks	ThermoFisher Scientific	156499
Tris HCL pH 8.5	Fisher Scientific	BP152-1	Store at 4°C
Ultrasonic bath	VWR	97043-960
Urea	Bioshop	URE001.1