In case you use a plate reader, you would be able to recalculate absolute numbers from the actual volume used for analysis (just in case your stopping gate is set to an unreachable number of gated cells). The only other possibility to obtain absolute cell numbers on BD cytometers is the use of a defined number of calibration bead (e.g. 25,000 blank Sphero Beads from BD), set your stopping gate in SSC/FSC on the beads (1/5 of total beads used) and recalculate.

I consider using volumetric analysis to calculate absolute cell number much less accurate than using calibration beads. Thus, I advise using beads. If you are trying to publish the results, I would say you cannot get around using the beads.

My biggest concern with using volume are the assumptions of a completely uniform cell suspension and no bubbles. The latter is very difficult to accomplish, considering one must get as close as one can to the former just to flow the cells without problems.

I would also suggest beads, although never used them so far. Moreover, how about any data analysis software (e.g. FlowJo)? After you gate the population you have, cells are counted and data are provided below Flow charts. The catch is that it may not be 100% accurate, especially if you are running the samples relatively fast (>1000 counts/sec.).

So, beads again!!!

Have you used counting beads? Does LSR II tells you the volume acquired? Generally if the concentration of cells is too high the count is not precise.

to have absolute count, you have two options:

- You can count cells in your preparation before flow analysis.(Malassdez, Thomas cell count) or automatic count (Biorad have a automatic cell count) with viability in trypan blue

- You can add fluoreccent bead (Trucount BD, FLowcount by BeckmanCoulter). This solution is the more accurate.

Did you use Collegenase D and DNase I for digesting your spleen? You need to digest the target organ for having good counts of macrophages, DCs...

I also agree that using calibration beads and a ubiquitous marker such as CD45 is the better way to calculate absolute numbers of cells in flow cytometry assays.

If you do not have calibration beads another approach could be the use of a hematologic counter and using lymphocytes, monocytes etc as the primary parameter for calculating of the specific populations.

Definitely use calibration beads. Depending on your flow the manufacturer will have them and you just add the same volume as the cells you are analysing, mixing them is vital. Add the calibrated count to your protocol and then absolute counts are then reported. Flow Count or True count, BC or BD.

As the guys have said above you can calibrate your flow cytometer with the use of beads to give an accurate count of volume from a defined number of events. These beads are at an exact concentration so that after a certain limit of beads is set and run an exact volume of bead solution will have passed through the flow. The volume data measured by the flow should match that of the volume of beads passed. If not you adjust what the flow is telling you to what volume of beads has passed (volume of beads passed= number of beads/concentration of beads).

That is usually what is done when you want to publish such experimental data. If you are happy with just rough estimates for now you can just read off the volume of cells that has passed through the flow. Gate on your population and work out the concentration of cells (events recorded/volume recorded). With that concentration and a defined volume of cells to begin with you can work out the absolute cell number from the original or any given volume (=concentration x volume).

I agree with Chantal for the preparation of your sample before going to the FACS machine, then there is no way around the use of a known number of counting beads/sample. It is the only way, I know of, to accurately evaluate the fraction of your sample that got analyzed by the cytometer. There is a catch though: the beads will allow you to calculate the number of cells that were in your tube before going to the machine. If your counts seem weird, you might be losing cells during your staining steps, you should include an unstained sample with beads to read at the cytometer to evaluate cell loss during staining/washing steps.

Hope this is useful.

Hi Anirban

A lot of people here have, rightly, pointed out the use of Calibration beads so that issue is covered. What I would like to know first is:

1- what is the protocol you are using for splenocytes?

2- Have you counted the cells using a Newbauer chamber and Trypan blue?

3- Have you tried smearing the cells and staining them (with giemsa, for example) to see what type of cells you are actually getting?

These are all steps you need to perform before going to the Flow Cytometer and spend your antibodies and beads.

Cheers

If you have a total live cell count (typan blue and count them down the microscope) you can then use your percentages obtained from the flow cytometer to calculate your counts of cell types.

You should wight the spleen, then, count the total number of cells (by cell count of an aliquote) per spleen. Then, if you stained (as is usual) a known number of total cells (commonly a million). You can then calculated from the number of events in each gate.

Best for absolute counts is, using beads produced for this purpose in the same tube with your sample as described by others. But if you do not have beads at hand a rough method can be used to have an idea. Formula is: total cell count x macrophage% x percentage of antibody of interest divided by 10000 which would give you cells per microliter. But i understand that you are not happy with the macrophage counts, they would be few in numbers, in order to have better results with more sensitivity you need to count more number of cells at least 50,000 nucleated cells per tube.

My feeling is that you can get comparable absolute cell counts by either,

a) plating X number of cells per well prior to staining, reading the sample, calculate percentages of each sort of cell (Y), then multiply %Y times X for total number of cells of type Y in your sample

or,

b) use beads, like BD Countbrite beads, spiked at a known concentration. Stop collecting at however many events your like, then backcalculate cell number using number of beads collected. The equation to use would be included in the protocol that comes with the beads.

I like option A the best bc the beads can be a little bit heterogenous in FSC/SSC/brightness and I worry that they can sometimes look like cells.

We used to add 2.5x104 Callibrite beads (Becton Dickinson) to each sample, and acquire until 5x103 beads were collected (or more if convenient), and then you just multiply the #cells by 5. However, in the stock tube label it says that it contains ~ 2x10^9 beads/ml, so this ~ sign makes me think that you can't really be sure of the real concentration of beads that you use as reference, so this is a useful technique to compare samples between them, but not to have an accurate absolute count. To have the best aproximation to an absolute count the best way I can think of is to count with the traditional Newbauer's chamber.

I really appreciate the the guidance from all of you, the trucount beads did offer good solution. Thank you guys!!!

Hi, normally you would count your splenocytes before the staining procedure (this might be done also with your flow cytometer). Then you can calculate the percentages using these numbers. However, there are confounding factors like "dead cells", "doublets", or the threshold you use; it might also be useful to do a collagenase-digestion in order to get the total number of macrophages. To avoid having to many other cells you can do a "live gate" collecting only the data from your macrophage gate. If you want to do a faster acquisition (more concentrated cell suspension) you might create a threshold more specific for your subset.

Concerning the true count: if this should give you information about the count in the corresponding spleen (and not just in the sample you measure) you must assure that your preparation does not have steps which would have an impact or a set variation on the cell count. This is more difficult for spleen than for blood (for blood you just make a non-wash protocol with wholeblood). There is also a problem, that the acquisition of the events is sometimes not uniform (all events within the last seconds..). True count beads will not help for these problems. The more you dilute your suspension the less you will have such variation.

If the flow rate is more or less constant and your sample preparation is good controlled, you might just set a "time gate" ( using "FlowJo", to take events from a fixed time frame) and compared the counts in the sample.

one more suggestion - everything has been said about truecount beads, so I'll skip that. but your problem in the first place might have something to do with macrophages.

If you want to get macrophages and dendritic cells from the spleen, use of collagenase in the isolation protocol is required, otherwise you will loose a lot of these cells sticking to the connective tissue. this might be where your varying percentages come from in the first place

Have really tried counting cells by trypan blue method and later on deriving the absolute number from percentages but its labour intensive as suggested by Adri Tomic