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I am trying to differentiate human monocytes to M1 or M2 using GM-CSF or M-CSF. GM-CSF (50ng/ml) treated monocytes were attached and growing pretty well. however, monocytes were not attached and seems dying in M-CSF( 100ng/ml)-treated wells. We isolated human monocytes from PBMC using Pan monocyte isolation kit from Miltenyi Biotec by negative selection. 5x105 monocytes were seeded in 24-wells in 500ul DXF medium from Promocell. Does anyone know what cause the problem and how to differentiate monocytes using M-CSF? Thanks!

I'm currently treating nucleus pulposus (adherent cells in the intervertebral disc) with various concentrations of cytokines and analyze the ROS level induced. After harvesting cells from a 6 well plate and adding DCFH-DA at 4 °C for 30 min, I loaded the samples on BD Acurri C6 plus and got these results:

Q: How should I avoid cells from dying and get enough live cells reasonably to represent the overall situation? Could it be anything wrong with harvesting adherent cells? BTW C6 has a bad reputation for doing cell analysis, should I use another flow cytometer?

I am culturing CD4+ T cells isolated from CRISPR-Cas9 knockin mice (Platt et al. Cell 2014, 1559:440). Initial activation on anti-CD3/anti-CD28 coated wells for 2 days seems normal and cells get activated (increase in size on FSC/SSC plot). However, after day 3 those cells do not proliferate well and start dying, irrespectively whether they are left on anti-CD3/anti-CD28 coated wells or moved on non-coated wells with addition of IL-2. Does someone have advice how to keep these CD4+ T cells in culture?

Hi,

I'm having problems with my AAV transduction. I tried to infect primary cortical neurons with AAV9. I can see fluorescence signals 48h after transduction. But at 72h after transduction, almost all the cells were dying and the fluorescence became strange (more like a circle and could not see the axons).

I infect primary cortical neurons at DIV8 and change the media after 12h. The MOI is 10^6.

I've tried lentivirus as well, but I had the same problem :( Fluorescence was observed after 48h but cells died after 72h...

Suggestions on what could be the possible problem are highly appreciated. Thanks a lot!

Xuetong

Currently I am potting some potato plantlets in a university as they contaminated during the holiday.

The last time I used only soil, and that potato is at the point of dying with poorly developed roots, and by quick Google, some says the potatoes want fertile and well drained sandy soil, so I finally potted them with soil, perlite, a small handful of vermiculite and plenty of sand until the mixture looked a bit greyish.

I found the mixture dry rather quickly, and unfortunately I didnt keep a record of the exact mixture, to pot the remaining plantlets better, can you kindly share your soil recipes for your potatoes in the laboratory?

Hi All,

I am trying to modify BT474 with the retroviral system (pBABE with pCL-Ampho packaging vector system, anyhow it is challenging to get a single colony after antibiotics selection (puromycine). Initially, I tried with 150 mm culture dish, but now I am working in 6 well because of cell morphology.

The problem I am facing is, after starting with antibiotics selection, maximum cells are dying, but the cells are being alive; they are not proliferating after that and being static for weeks. So it's difficult to get the transfected cells.

If someone can suggest some relevant suggestions to get over this problem, it would be beneficial.

Thank you so much.

Hi all,

I have been using PHA as positive control of stimulation on both healthy and diseased PBMCs for about a year. 2.5ug/mL, 10e5 cells/well.

In the past few weeks, any cells I plate with PHA activate (shown as activation markers by flow) but also die in large quantities, visibly even after 24hr stim.

I haven't changed my protocol, re-made fresh culture media and PHA dilutions. Yet the cells keep dying when activated -even freshly isolated, healthy donor cells.

What else could be the cause?

I've been trying to activate and expand naive T cells that have previously been isolated from PBMCs with a pan-T cell isolation kit from Miltenyi and cryopreserved in 90% FBS 10% DMSO. I have decent viability post thaw >85% with ~10% loss in viability from the pre-freeze viability. However, over the 10 days that i'm expanding the T cells, I initially see good activation, but then the T cells start dying off and I end up with abysmal viability by day 10.

Here's the protocol i'm using:

I rapidly thaw the vial of T cells in the water bath and when the vial is almost completely thawed, add 1mL T cell media dropwise. I transfer the full volume to a 15mL tube with more T cell media dropwise, then spin at 300xg for 5 minutes. I activate the T cells in Miltenyi TexMACS medium supplemented with 300 IU/mL IL2 and CD3/CD38 Dynabeads at a 1:1 T cell:bead ratio. I plate the cells at 2 million/mL in a 24 well plate for the initial activation. I expand the T cells for 10 days, checking the viability every 2 days and replenishing media/cytokine if the media starts looking yellow, keeping T cells between 1.5-2 million/mL.

This protocol has worked very well for me from T cells isolated from freshly prepared PBMCs, but hasn't been working well with the cryopreserved T cells. I initially see very good clumps forming that get larger, but towards the second half of the 10 days the clumps don't get very large and the T cells start dying. Over 10 days I had less than a 2-fold expansion, whereas with fresh T cells I could get a 4-5 fold expansion over 10 days.

Does anyone have experience activating and expanding thawed naive T cells or have thoughts on why my T cells may be dying? Whats weird is that I've used the exact same vial of frozen naive T cells to generate CART cells using a 3 day Dynabead activation with IL7 & IL15 followed by viral spinoculation, and the viability of these CART cells is fine after a few days of expansion after spinoculation.

May 12th, 2011 at 1:28 pm

That evil Empire needs to be snuffed out. When they come onto my turf they must know that they are going to be in a fight. (background music cues up)

When you’re a Jeti you’re

a Jeti all the way.

From your first cigarette to

your last dying day.

When you’re a Jeti,

If the spit hits the fan,

You got brothers around,

You’re a family man!

You’re never alone,

You’re never disconnected!

You’re home with your own:

When company’s expected,

You’re well protected!

Then you are set

With a capital J,

Which you’ll never forget

Till they cart you away.

When you’re a Jet,

You stay a Jeti!

Hi,

 I'm using mouse embryonic stem cells. Upon certain condition I found that cells are dying. They become shrinked and floating around. So I just want to know whether these cells are undergoing apoptosis. I bought TUNEL assay kit from Roche, but I can't decide when should I prepare my samples.

Q1. Do I have to collect both floating (dead) and adherent cells? When I first tried only with adherent cells while washing out all the floating cells, adherent cells are barely stained while some leftover floating cells are stained very well. I know that cell debris sometimes show fluorescence, so I cannot be sure that floating cells are really 'stained' or just showing artificial signals.

Q2. Can floating cells be fixed? I'm doing TUNEL assay using slides and detect the signal under fluorescence and/or confocal microscope. If only floating cells show TUNEL signal how can I fix the cells? Do you think I have to use FACS instead?

Q3. It might be related question. Can cells be TUNEL positive (and dying?) and adherent at the same time?

Thank you so much!