Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

Published: August 31, 2023

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…

Divulgaciones

The authors have nothing to disclose.

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

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Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood. J. Vis. Exp. (Pending Publication), e21564, doi: (2023).

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