Three methods for isolating neutrophils

1、 Standard method: Ficoll Hypaque density gradient and red blood cell lysis method

(1) Take a 50ml polyethylene tube and collect sterile human peripheral venous blood. Add 4.4ml of 3.8% or 5% citrate solution to a volume of 40ml. The above whole blood is 300gX20min, centrifuged at room temperature.

(2) Suck out the platelet rich supernatant, 2500g x 15min, and prepare platelet free plasma (PPP).

(3) Add 5ml of 6% Dextran (molecular weight 500000, prepared with sterile physiological saline) to the remaining settled whole blood, and adjust the final volume to 50ml with physiological saline (0.9%), gently and thoroughly mixing. Settle at room temperature for 30 minutes.

(4) Suck out the upper layer of fluid rich in white blood cells, 275gX6 minutes.

(5) The precipitated cells were resuspended with 8ml of PPP physiological saline (1:4) and transferred to a 15ml centrifuge tube.

(6) Add 3ml Ficoll Hypaque (density 1.077) to the suspension cells, 750g X for 5 minutes, at room temperature.

(7) Suck neutrophils and red blood cell layers, resuspend cells with 0.155M NH4Cl to lyse red blood cells, then wash neutrophils twice with equilibrium solution containing 0.25% BSA Hanks (HBSA, calcium free), and finally resuspend with HBSA (calcium containing).

(8) More than 95% of neutrophils can be obtained using this method.

2、 Discontinuous density gradient plasma Percoll centrifugation method

(1) Prepare Percoll storage solution: The volume ratio of Percoll stock solution (100%) to 0.9% NaCl is 9:1.

(2) Take a 50ml polyethylene tube and collect sterile human peripheral venous blood. Add 4.4ml of 3.8% or 5% citrate solution to a volume of 40ml. The above whole blood is 300gX20min, centrifuged at room temperature.

(3) Suck out the platelet rich supernatant, 2500g x 15min, and prepare platelet free plasma (PPP).

(4) Add 5ml of 6% Dextran (molecular weight 500000, prepared with sterile physiological saline) to the remaining settled whole blood, and adjust the final volume to 50ml with physiological saline (0.9%), gently and thoroughly mixing. Settle at room temperature for 30 minutes.

(5) Suck out the upper layer of fluid rich in white blood cells, 275gX6 minutes.

(6) The precipitated cells were resuspended with 2-3 ml of PPP.

(7) Add 2ml 42%, 2ml 51% Percoll (all freshly prepared with PPP), and 2-3ml of cell suspension to a 15ml centrifuge tube for 275gX10min.

(8) Mononuclear cells and some platelets are located between plasma and 42% Percoll solution, while neutrophils are located between 42% Percoll and 51% Percoll. Platelets can be removed by centrifugation with 25% Percoll for 5 minutes, or a third 25% Percoll can be added to the original density gradient for centrifugation together.

(9) Wash the neutrophil layer once with PPP solution, and then wash once with Krebs Ringer phosphate buffer containing sugar (KRPD, pH 7.23).

(10) By using this method, 80% neutrophils can be obtained with a purity of over 95%.

3、 LPS Free method

(1) The method of using deformable cells (such as white blood cells) for lysis has shown that containers and solutions do not contain LPS, meaning that the content of LPS is less than 0.1ng/ml.

(2) Collect sterile venous blood and anticoagulate with 10% sodium citrate.

(3) Add 6% Dextran (mol wt 70000) to anticoagulant in a ratio of 3:1 (vol/vol, blood/dextran) at room temperature for 1 hour.

(4) Collect plasma from white blood cells, centrifuge, and remove supernatant.

(5) Red blood cells are lysed with low osmotic 0.2% NaCl for 15 seconds (this step can also be replaced by 0.155M NH4Cl for 15 seconds).

(6) Immediately add 10ml of 1.6% NaCl (containing 2mg/ml of sugar) before centrifugation.

(7) Wash twice with KRPD after centrifugation.

(8) This method obtains the number of neutrophils similar to Method 2, but with a purity of only 80-85%, with the remaining being lymphocytes and monocytes.

4. Purification of neutrophils obtained from 3

(1) Because Method 3 can only obtain neutrophils with a purity of 80-85%, Method 2 and Method 3 can be combined to purify neutrophils.

(2) Collect plasma rich in white blood cells obtained from method 3 dextran sedimentation, 275g * 6 minutes, and centrifuge.

(3) Resuspend 2ml of PPP for bottom sedimentation.

(4) Add 2ml 42%, 2ml 51% Percoll (all freshly prepared with PPP), and 2-3ml of cell suspension to a 15ml centrifuge tube for 275gX10min.

(5) Wash once with PPP and once with KRPD respectively.

(6) The purity of neutrophils obtained by this method is over 99%, and there is no intermediate step of red blood cell lysis.

Preparation of Krebs Ringer buffer (KRPD buffer):

130 mM NaCl, 5mM KCl, 1.27 mM MgSO4, 0.95 mM CaCl2, 5 mM glucose, 10 mM NaH2PO4/Na2HPO4, pH 7.4

Preparation method:

A solution: NaCl 7.5985g; KCl 0.3727g; CaCl2 0.1054g, glucose H2O 0.9495g, dissolved in 100ml of ddH2O;

B solution: NaH2PO4-2H2O 0.2964g Na2HPO4-12H2O 2.9011g, dissolve in 100ml of dH2O, then add MgSO4-7H2O 0.3130g to fully dissolve.

Mix A and B, adjust the volume to around 990ml with ddH2O, adjust the pH to 7.2-7.4 with 1M NaOH or 1M HCl, and make up to 1000ml.