We want to check if we can inhibit Net in Neutrophils that was induced by PMA with our inhibitor. However, we saw that we are loosing up to 70% of the cells within a few hours even without any PMA treatment.
In this experiment we have incubated Neutrophils on Poly-L-Lysin slide for 30 minutes. Washed them (gently) 3 times and incubated for 1 hour (with RPMI). At that point there was a loss of 35%.
After fixation (Methanol 100% for 2 minutes) and washes we have lost up to 70% from the initial number.
Is that reasonable?
Do anybody know how to prevent this loss of cells?
Thanks
Of note, experiments with other slides (utilizing cytospin) didn't yield better results.
Hello,
Neutrophil loss is normal as these are end-differentiated cells. We usually find that over 50% are apoptotic after 24 hours in culture after isolation from blood. They become apoptotic even more quickly if they are handled too roughly or exposed to media that tips them into cell death more readily.
The main thing that seems to really tip them into apoptosis is the isolation technique used to isolate neutrophils. Dextran sedimentation of blood causes apoptosis in neutrophils if the dextran is left with blood for more than 30 minutes. Spinning neutrophils on a Ficoll gradient at colder temperatures than ambient (21 C) also increases neutrophil apoptosis. Finally, handling neutrophil preparations too roughly by even moderately vigorous pipetting causes increased cell death.
We found the best possible neutrophil isolation kit is the MACSxpress one from Miltenyi Biotec. It does not require dextran or Ficoll for preparation, and can purify neutrophils by negative selection directly from blood. The downside is that this is an expensive kit to use. But we have had perfect preparations of neutrophils using this kit.
When we adhere neutrophils to glass coverslips for imaging, we just use 95% ethanol-washed and air-dried 12 mm No. 1 glass coverslips, with no poly-L-lysine coating. We found there was no difference with or without poly-L-lysine for optimal adherence. Neutrophils adhere readily to clean glass in serum-free RPMI after 15-30 min at 37 C in a humidified incubator. The key is to be sure that the media is free of proteins, including serum.
Good luck!
Best,
Paige
Hi Miron,
I have often grown these cells on plain glass coverslips (sterile) in 6-well plates and have never had a problem with detachment, so I don't understand these losses.
One thing that occurs to me as a possible cause is the extremely short (30 minute) time for the cells to adhere to the slides; perhaps I am misunderstanding you. I would let them settle down for at least 12-16 hours before starting.
Have you tried a trypan blue dye exclusion assay before (and after) PMA? Have you also checked for mycoplasma contamination? You might like to try 2% formaldehyde as a fixative (5ml 38% formaldehyde + 95ml PBS) after first washing gently with plain PBS; 10 minutes is enough. Provided you are very gentle with your washes, there should be virtually no losses.
I hope this helps.
Best wishes,
Simon
I agree with Simon, there really shouldn't be any significant losses. I have also never needed poly-l-lysine, neutrophils (and monocytes) stick to glass and most plastic very well without any further aids. I find that 2 hours is plenty to allow them to adhere and if you are testing differences in NET formation in response to some drug or other you can do that quite effectively without the initial wash - i.e. just treat them with PMA and/or your substance of choice.
Good luck.
Hi Miron,
Like Simon, we use untreated glass coverslips, 12 mm diameter which fit nicely into 24-well plates. In our hands even untreated cells adhere well, we do not encounter notable cell loss. We avoid polylysine since it can induce cell death. Normally the experiments last between 30 min and 5 hrs after seeding the cells, fixation with 2% paraformaldehyde in PBS. Change of media very gentle at the periphery of the coverslip.
Best wishes, Volker
Hello,
Neutrophil loss is normal as these are end-differentiated cells. We usually find that over 50% are apoptotic after 24 hours in culture after isolation from blood. They become apoptotic even more quickly if they are handled too roughly or exposed to media that tips them into cell death more readily.
The main thing that seems to really tip them into apoptosis is the isolation technique used to isolate neutrophils. Dextran sedimentation of blood causes apoptosis in neutrophils if the dextran is left with blood for more than 30 minutes. Spinning neutrophils on a Ficoll gradient at colder temperatures than ambient (21 C) also increases neutrophil apoptosis. Finally, handling neutrophil preparations too roughly by even moderately vigorous pipetting causes increased cell death.
We found the best possible neutrophil isolation kit is the MACSxpress one from Miltenyi Biotec. It does not require dextran or Ficoll for preparation, and can purify neutrophils by negative selection directly from blood. The downside is that this is an expensive kit to use. But we have had perfect preparations of neutrophils using this kit.
When we adhere neutrophils to glass coverslips for imaging, we just use 95% ethanol-washed and air-dried 12 mm No. 1 glass coverslips, with no poly-L-lysine coating. We found there was no difference with or without poly-L-lysine for optimal adherence. Neutrophils adhere readily to clean glass in serum-free RPMI after 15-30 min at 37 C in a humidified incubator. The key is to be sure that the media is free of proteins, including serum.
Good luck!
Best,
Paige
Very nice answer Pagie, I agree with everything you said. The manipulation with blood during isolation of cells PMN or Ly is very tricky and only a slight change/ mistake in protocol (like temperature during centrifugation) can induce cell apoptosis.
My experience in the case of Ficoll isolation is that at least two centrifugation (10 min each) increase the number of viable cells. I think that the Ficoll remains after only one centrifuge step and cause the death (more than 30%).
I used all 3 possible coated slides from ibidi for microscopy (Poly-L-lysin, collagen and ibitreat) and only the uncoated slide was not pre-activating my cells.
check if that is also possible for your experiment
bests
Hello,
Just another tip.. As per your description a significant percentage of the cells is lost after incubation and fixation steps, both including washing the slides. I have previously worked with WBC, mainly lympho, but isolation is never perfect and neutrophils were also present. In those experiments we attached cells to slides using a cyto-centrifuge at 800 rpm, and after several intense washes (required by the experimental protocol we used) we still observed a good cell density attached to slides, including neutrophils.
Perhaps using uncoated slides and cyto-centrifuge could reduce the loss of cells in your case. Hope to be of some help.
Hello,
There's a very recent paper on prolonging neutrophil viability in case anyone is interested:
http://www.bloodjournal.org/content/128/7/993?sso-checked=true
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