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…
開示
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 |
