Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

Abstract

Source: Sionov, R. V., et al., Isolation and Characterization of Neutrophils with Anti-Tumor Properties. J. Vis. Exp. (2015)

In this video, we demonstrate the isolation of cytotoxic high-density neutrophils and low-density neutrophils from the blood of a tumor-bearing mouse by discontinuous sucrose gradient centrifugation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Neutrophil Isolation Isolation of cytotoxic neutrophils from blood of tumor-bearing mice. Dilute 1 ml of blood drawn from a tumor-bearing mouse in PBS containing 0.5% (w/v) bovine serum albumin (BSA) to a final volume of 6 ml. Fractionation of diluted blood on a freshly prepared d…

Representative Results

Figure 1. Neutrophil purification from whole blood. (A) 3 ml of 1.077 g/ml sucrose is carefully layered on top of 3 ml 1.119 g/ml sucrose to form a discontinuous gradient. Whole murine blood, diluted in PBS-BSA (0.5%) to a final volume of 6 ml, is then layered on top of the 1.077 g/ml sucrose. (B) Following a 30 min spin at 700 x g with no break, 3 distinct fractions can be observed; R – red blood…

Materials

Mouse 4T1 breast carcinoma cells	ADCC	CRL-2539	Growth medium: DMEM + 10 % heat-inactivated FBS
24-well Tissue Culture Plate	Falcon	353047
100 mm Tissue Culture Plate	Corning	430167
25 cm2 Tissue Culture Flask	Nunc	156340
90 mm Bacterial Grade Culture Dish	Miniplast, Ein Shemer, Israel	20090-01-017
15 ml Sterile Conical Centrifuge Tube	Miniplast, Ein Shemer, Israel	835015-40-111
50 ml Sterile Conical Centrifuge Tube		835050-21-111
Falcon 12×75 mm Round-Bottom Polystyrene Tube	Becton Dickinson	352058
HPLC grade water	J.T. Baker	4218-03	Autoclave
Histopaque-1119	Sigma	11191	Sterile filter through a 0.2 mm filter.
Histopaque-1077	Sigma	10771	Sterile filter through a 0.2 mm filter.
PBS+0.5% BSA			Dissolve 2.5g BSA in 500 ml PBS, and sterile filter through a 0.2 mm filter.
PBS+1% BSA			Dissolve 1g BSA in 100 ml PBS, and sterile filter through a 0.2 mm filter.
Bovine serum albumin (BSA), fraction V	Sigma	A7906