Hello,

To isolate neutrophil, I use a percoll gradient 60% to 80%.

1. Red blood cells Lysis --> diluate your WBC in 1mL of HBSS-/- (without Mg and Ca) + SVF 5%.

2. diluate Percoll 9mL of Percoll with 1mL of NaCl 1,5M to do a 100% of Percoll solution

2. Diluate :

- 4mL of Percoll 100% with 1mL of HBSS1X --> Percoll 80% solution

- 3mL of Percoll 100% with 2mL of HBSS1X --> Percoll 60% solution

3. Drop off slowly the Percoll 60% solution on the Percoll 80% solution

4. Deposite your 1mL of WBC on the top slowly and centrifugate 3000rpm during 10min without break --> You will have on top monocyte and lymphocyte ring and under a neutrophil ring.

I perform a wash and then diluate in HBSS+/+ (with Ca and Mg) + SVF to perform stimulation experiment (NETosis).

Good luck,

Geoffrey

Although we did not use Percoll in this study, but Ficoll-Hypaque instead here is our protocol for neutrophil isolation and labeling (Eur. J. Pharmacol., 2003, 477: 261-7)

"For each experiment, granulocytes were isolated from

heparinised venous blood of a healthy, non-smoking volunteer

with no known existence of an inflammatory disease.

All volunteers gave informed consent and the study was

approved by the local Ethics Committee. Granulocytes were

separated from mononuclear cells by density centrifugation

over Ficoll-Hypaque, d = 1.077 g/ml for 25 min, 1000g at

room temperature. Contaminating erythrocytes were removed

from the granulocyte suspension by isotonic ammonium

chloride lysis at 0 jC (Roos and Loos, 1970) and

subsequent centrifugation. Isotonic shock solution contained

155 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, and 10

mg/l of phenolred. The pH of the solution was adjusted to

7.40 at 0 jC. After centrifugation, the granulocytes were

collected, washed twice in PBS, and suspended in PBS at a

concentration of 107/ml.

To each millilitre of granulocyte suspension, a volume

of 5 Al calcein-AM (5 AM in DMSO) was added,

followed by incubation at 37 jC for 15 min. After

uploading was stopped by adding excess PBS, not incorporated

label was removed by washing the cells twice in

HBSS (400g, for 10 min at room temperature). Afterwards,

the cells were resuspended in HBSS/0.1% bovine

serum albumin at a concentration of 2106 cells/ml and

incubated for 1 h at 37 jC in humidified air with 5%

CO2 to activate the label."

Best regards,

Frans van Overveld

Geoffrey Garcia Thank you so much for your protocol.

Could you clarify a little bit more about your protocol:

1. Usually, how much is the amount of blood you will take for each sample? And after collect the samples, you could keep your samples in 4 degree fridge for how long before start isolation?

2. dilute Percoll 9mL of Percoll with 1mL of NaCl 1,5M to do a 100% of Percoll solution

--> Could I dilute Percoll in HBSS instead?

My lab is using Percoll PLUS (Cytiva)

2.Deposite your 1mL of WBC on the top slowly and centrifugate 3000rpm during 10min without break

--> some protocol I know mentions centrifugation in 20mins, do you think it is ok with longer time of centrifugation?

3.Could you tell me more about the wash step?

4. I need to keep neutrophils alive, maybe several hours at least after treating with LPS just to check some gene expression.

So do you think medium of HBSS+/+ (with Ca and Mg) + SVF is enough for the survival? Actually, I'm thinking of using cell culture medium as RPMI + FBS +P/S as normal cell culture protocol.

I look forward to your response.