Hello everyone,
I'm doing a cytotoxic assay by labeling my target cells with Calcein AM, cocultured with different ratios of effector cells. However, my result is still a mess and unexplained, the higher ratio has the lower value.
Please check my procedure below :
1. Prepare Target cells : 10^6 cells/1mL HBSS free
2. Add Calcein AM to the buffer in a final concentration of 15µM for fluorescent labeling, mix well
3. Incubate 37oC for 30min with occasional shaking
4. Wash cells 2 times with HBSS free
5. Suspension 5x10^3 target cells in 100µL HBSS free, plating in 96-well, U bottom microtiter plates (triplicate wells/samples)
6. Scan prior to incubation to detect pipetting errors
7. Suspension CTL in different ratios: E:T =80:1,40:1, 20:1, 10:1, 5:1, 2.5:1
8. Incubate 37oC for 4hours
9. Centrifuge plate 1000g, 5min, move supernatant to new plate (75-100µL) (Costar black flat plate)
10. Scan the plate and fluorescence is quantifed, 490nm excitation and 520nm emission wavelength
11. Percent specific lysis = (experiment release-spontaneous release)/(maximum release- spontaneous release) x100
Spontaneous release: target in media alone
Maximum release: Target cells lysed in medium plus 2% Trixton X-100
Each measured in at least 6 replicate wells
Calcein AM : Molecular Probe , 1mg powder, dilute in DMSO, briefly calculate : 1mM add 15µL into 1mL to reach final concentration 15µM
Machine : TECAN Infinite Pro 200
Thank you so much.
Thank you Bhagat Patlolla, but you mean I should use RPMI free during Calcein staining, or 4 hours coculture target and effector cells?
One question - do your positive (TX-100) and negative (just target) controls behave as expected? Also if it is possible I would try to run assay in flat bottom 96 well plate and check it on fluorescent microscope at step 6 to see if the cells are properly stained and after step 8 to see if they are actually dying.
Thank you Pavel Ostasov,
My positive control always show similar value and very high compare to other sample, whereas negative control sometimes show the higher value than samples (at low ratio of E:T). For example, positive control was 750, negative could be 115-130, samples at ratio 5E:1T was 107-112 -116.
When I read a very old publish (2004), they explained that, this phenomenon may due to the spontaneous release of Calcein and subsequent UPTAKE by effector cells.
I will try to scan fluorescence after step 6. However after step 8 , if I don't centrifuge and transfer supernatant to another plate, it's impossible to scan the plate because there are so many effector cells can affect on the result (too much high to read).
Thank you.
Hi Paulo,
One of my lab's members is using Calcein-AM and PI to check the cell dead, however my experiment is different, I wanna check the Calcein released into supernatant due to cell lysis. I think maybe you mentioned to Live/Dead asay whereas mine is Cytotoxic assay.
Thank you.
Bahgat is right. Do not use FBS with calcein, use plain RPMI, PBS, HBSS, etc. There are components in the FBS that will cleave the AM group off the calcein-AM, causing it to fluoresce outside your cells. This causes low signal-to-noise ratios and gives wide variation in your results.
Hi everybody,
My last result is still very confused. In the attached file, they also stained the target cells in serum-free culture medium containing Calcein for 30 min (page 696, figure 5D)
However, they also resuspended target cell in PBS containing 5% FCS after staining (page 691). So I really want to ask in more detail
1. We just stain Calcein in serum free media/ HBSS/PBS but we still use FBS/FCS during co-culture process (must reduce 10% serum --> 5%)?
2. Can I co-culture for longer time? for example 6-8-10 hours? Or increasing the Effector: Target ratio is the better idea?
Sincerely,
you better check your target cells whether they are MRP-overexpressed cell lines. Calcein retention is a test to check whether a particular cancer cell line expressed MRP (multidrug-resistant protein). If a cancer cell line expressed MRP, calcein can be easily efflux out to medium. Therefore, you have less intracellular calcein retention and more calcein in medium. The gap between maximal release and spontaneous release is very close..and the assay is very sensitive at this point..try other cytotoxicity assay like the CytoTox-Glo™ by promega..i havent try..but at least it depends on protease release from dead cells..
Hi, I'm facing almost similar problem with you. I'm wondering whether you manage to solve this problem. Could you share with me you experience of this assay? Thanks.