Ficoll-右旋糖酐法提取中性粒细胞
1.采集10mL全血于EDTA预处理过的试管中，用不含钙和镁离子的1x HBSS缓冲液稀释之，比例为1:1。采集的样本必须尽可能早地进行分离。
2.提前另取一50ml离心管，加入与全血量相等体积的Ficoll-Paque溶液。向Ficoll溶液上方缓慢注入稀释过的全血，注意不要破坏液面。
3.立即600g室温下9:2减速离心15分钟(即ACC-9，DEC-2)。
4.离心后可观察到血浆、外周血单核细胞(PBMC)、Ficoll-Paque溶液和含有粒细胞的红细胞沉淀四层。前三层弃掉，以获得第四层红细胞和中性粒细胞混合物。
5.立即将混合物重悬于1×HBSS缓冲液5ml中，以进行进一步处理。
6.红细胞和粒细胞悬浮液立即与等量3%右旋糖酐混合，室温避光孵育30min。 7.收集上层富含中性粒细胞的上清液，弃去沉淀。500g室温下离心10min。 8.弃上清。最后用3倍全血量的红细胞裂解缓冲液裂解红细胞，裂解时间10分钟。 9.加入等量1 x HBSS缓冲液终止裂解。400g室温下离心10min。
10.离心后弃上清，立即用1ml含10%FBS的RPMI1640完全培养基重悬沉淀，即得中性粒细胞悬浮液。
11.（可选）镜下对中性粒细胞计数，以确定后续实验方案。
12.（可选）CD45、CD16、CD66b抗体染色后，流式测定中性粒细胞的细胞数和纯度。
注：
1、全过程中的1×HBSS可用无菌生理盐水代替，量是一样的，这点有文献佐证。但如果用HBSS要订购w/o Mg2+ & Ca2+的，这种相比于生理盐水来说优点是不会刺激中
性粒细胞导致其活化（活化的后果是什么也不是特别清楚…炎症因子表达上调？）。
2、3%右旋糖酐用无菌生理盐水配制。
3、(2)、(3)步骤衔接要快，否则分层效果会比较差。
4、(11)和(12)可能是很重要的，因为每一次能提取的细胞量都因实验误差及个体异质性而不同，如果需要严格控制共培养细胞数目比例的话，这一点必不可少。
5、与肿瘤细胞共培养的比例有待摸索。文献用5×106肿瘤细胞与100×106血小板共培养，而血液中血小板（100-300×109/ml）相对于中性粒细胞（4-10×109/ml）之比例浮动于10-75:1之间，故设计共培养的比例目前暂定1×、2×、5×、10×、20×，待免疫荧光、细胞凋亡、细胞周期等实验结束后再选择合适比例做后续实验。
6、步骤(8)中3倍红细胞裂解液，这个体积个人觉得可以再少一点，目前台上一管血8-9ml（实际收不到10ml），如果(8)、(9)都是3倍量，50ml的管子可能撑不住。
7、流式方法：将所取细胞于500g条件下5min离心（最好是减速离心，但实际操作中不减速好像伤害也不大，注意是g不是rpm），弃上清后加入PBS重悬，再同样条件下离心，反复三次，再弃上清后，用300-400μL 1x binding buffer（凋亡试剂盒中有这个，实验室没有的话用PBS也行）重悬，立刻加入5μL CD45抗体，避光孵育30分钟。随即在流式细胞仪中测定纯度。如果顺便想测凋亡的话，可在CD45避光孵育完后再按凋亡试剂盒步骤加入Annexin V FITC/PI，避光孵育15分钟测流式。
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简易提取法： PROTOCOL
NOTE: All experiments were conducted in accordance with local institutional ethical guidelines.
1. Blood Drawing
1. Through antecubital venipucture, collect 14 tubes of blood from a healthy volunteer into green top (heparinized) tubes and invert each tube before settling on ice. Collect blood within 10 - 15 min of experiment to ensure optimal neutrophil yield.
2. Neutrophil Isolation from Whole Blood
1. Wash each green top tube of blood with 5 ml of PBS without Ca and Mg to dilute the blood. In each of 4 x 50 ml conical tubes, add 15 ml of Lymphocyte Separation Media (LSM) at RT. Using a 18 G needle mounted on a 60 ml syringe, carefully layer the diluted blood onto the LSM, creating a sharp LSM-blood interface.
2. NOTE: Avoid mixing of the layers as much as possible.
3. Centrifuge at 800 x g for 30 min at 21 °C without break.
4. NOTE: Sudden breaks can cause mixing of the different layers.
5. Observe the erythrocytes and neutrophils sediment at the bottom. Aspirate and discard the top 2 layers (plasma and LSM) making sure to get rid of the interface between these layers which contains lymphocytes and mononuclear cells, leaving only the bottom red layer.
6. To each tube, add 20 ml of Phosphate Buffer Saline (PBS) and 20 ml of 6% Dextran solution. Gently invert each tube to mix with the layer of erythrocytes and neutrophils and allow to sit at RT for 30 min.
7. NOTE: This will allow red blood cells (RBCs) to sediment at the bottom.
8. After 30 min, transfer the neutrophil rich supernatant into fresh tubes and discard the RBC pellets. Centrifuge supernatants at 450 x g at 4 °C for 5 min. After centrifugation, discard supernatant. The pellet will contain mostly neutrophils and few RBCs.
9. Prepare a lysing solution by adding 0.5 ml of lysing buffer to 4.5 ml of sterile water. Lyse remaining RBC with the prepared 5 ml of lysing solution, transferring all resuspended pellets into one tube. Allow lysis at RT in the dark for 10 min.
10. Centrifuge at 450 x g at 4 °C for 5 min. Discard supernatant and wash pellet with 5 ml PBS without Ca and Mg and centrifuge again at 450 x g at 4 °C to get rid of any remaining lysing solution. The pellet obtained at this point contains the neutrophils. Resuspend the pellet in 30 ml of cold 3% RPMI and put on ice.
11. NOTE: 3% RPMI is made by supplementing RPMI media with 3% Fetal Bovine Serum (FBS)
12. Verify purity of the sample using Methylene blue staining. >95% of cells
should be granulocytes with multi-lobar nuclei. Use Trypan blue staining to verify >95% viability of cells and count the final neutrophil yield. Dilute neutrophils in ice cold 3% RPMI to obtain a final concentration of 5 x 106 neutrophils/ml.
3. NETs Generation
1. Stimulate neutrophils with 500 nM of PMA (per 30 ml of neutrophil solution) and incubate on a 150 x 25mm flat tissue culture dish with 20 mm grid for 4 hr at 37 °C 5% CO2. This will allow NETosis.
2. After 4 hr of stimulation, gently aspirate and discard the media, leaving the layer of NETs and neutrophils adhered at the bottom. Do not disrupt this layer.
3. Using a total of 15 ml of cold PBS without Ca and Mg per dish, wash the bottom of each dish by pipetting 15 ml of PBS on the bottom of the dish in order to lift off all adherent material from the bottom.
4. Collect solution obtained from washing each dish (step 3.3) in a 15 ml conical tube and centrifuge for 10 min at 450 x g at 4 °C. Neutrophils and any remaining cells will pellet at the bottom, leaving a cell-free NET-rich supernatant.
5. Divide supernatant into 1.5 ml micro-centrifuge tubes and spin for 10 min at 18,000 x g at 4 °C. This will allow all DNA to pellet.
6. NOTE: Centrifugation in larger tubes is easier and less time consuming if such high speeds are attainable on the regular centrifuge, otherwise will need to divide supernatants in smaller tubes in order to use high speed micro-centrifuge.
7. Discard supernatant and resuspend all pellets obtained together in ice cold PBS to a concentration corresponding to 2 x 107 neutrophils per 100 μl of PBS. This will yield the cell-free NET stock that can be used for subsequent experiments.
8. Measure DNA concentration in the sample obtained using spectrophotometry or alternate DNA quantification tool. An adequate concentration in the sample should range between 140 - 180 ng/μl.
4. NET-Cancer Cell Static Adhesion Assay
1. Add 100 μl of the previously obtained NET stock per well in a 96 well flat bottom plate and allow to coat wells O/N at 4 °C in the dark.
2. Between 12 and 20 hr later, verify formation of a uniform monolayer of cell free NETs at the bottom of the wells under the microscope (Figure 1). At this point, gently aspirate all non-adherent material out of the wells, making sure not to disrupt the NET monolayer at the bottom.
3. Add 100 μl of 1% Bovine Serum Albumin (BSA) blocking solution to each well and leave for 1 h at RT.
4. Detach A549 cancer cells from a T-75 flask of using 2 ml of 0.25% Trypsin solution. Once detached, add 10 ml of A549 media to trypsinized cells and centrifuge at 450 x g at 4 °C for 5 min. Discard supernatant and resuspend cells in media to obtain a concentration of 2 x104 cancer cells per 100 μl media.
5. NOTE: A549 cells were grown separately. Briefly, cells were cultured and maintained in DMEM F12 media containing 10% FBS and 1% Penicillin
Streptomycin and incubated at 37 °C 5% CO2. Once 70 - 80% cell confluence was reached, they were detached using 0.25% Trypsin-EDTA and resuspended in the same media described above.
6. Stain cancer cells using CFSE by adding 1 μl of CFSE per ml of media and leave to stain at RT for 10 min. After staining, centrifuge down at 450 x g at 4 °C for 5 min then discard supernatant and resuspend cells in initial volume of media to obtain a concentration of 2 x104 cancer cells per 100 μl media.
7. After 1 hr of NETs blocking, gently aspirate blocking solution and add 2 x104 cancer cells in media, which is equivalent to 100 μl, per well over the NET monolayer and allow to adhere for 90 min at 37 °C 5% CO2. Gently aspirate the cells and add 100 μl of PBS to each well to wash any non-adherent cells.
8. In some wells, add 1000U of DNAse1 per well for 10 min before washing to degrade NETs. This will serve as negative control. In other wells, add 100 μl of sterile water per well for 10 min, which will serve as the vehicle control (VC).
9. NOTE: 3 replicates per condition are usually performed to increase sample size.
10. Aspirate and discard all solution in the wells leaving only NETs and adherent cancer cells at the bottom. Add 100 μl of 4% formaldehyde solution per well to fix adherent cancer cells to NETS and read assay under the fluorescence microscope (Figure 1). Plot and analyze the results (Figure 3).
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