plate bound Anti-CD3 Anti-CD28 protocol

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mforrester's picture
plate bound Anti-CD3 Anti-CD28 protocol

Hi, I am planning on stimulating T cells with plate bound anti-CD3 and anti-CD28 I know I want to use a concentration of 1ug/ml but I'm having issues finding a protocol to do this.  What do I use as a buffer for coating? what do I wash with? how long should I incubate the plate for? and at what temp?

I'm sure that lack of protocols is due to its common use in labs and therefore no need to publish it but my lab has previously used the beads rather than coated plates and therefore I have no source of this protocol near by. 

any info would be a huge help, thanks

samm's picture
I usually use NA/LE or LEAF

I usually use NA/LE or LEAF abs from BD Pharmingen, eBioscience or BioLegend (clones 145-2C11/37.51 for mouse). The two protocols we have for even coating both call for an overnight incubation, but shorter incubations may work.
Use either bicarb buffer at pH 8.8 or PBS to make a 1 ug/ml solution of aCD3. 0.5 ug/ml aCD28 can be either added to plate, or added in a soluble manner. (All mouse, and most human CD28 mAbs stimulate rather than block, so a soluble presentation works pretty well). The aCD3 soln is added at ~100 ul/well, place the lid, and seal with parafilm. Keep flat in a refrigerator o/n. Next morning, aspirate out Abs, add equal volume of culture medium with FBS, incubate at RT for ~10 mins, aspirate medium (do this 2x), and finally replace with medium+cells+experimental treatment - without ever allowing the wells to go dry (do this in stages if necessary - 16 or 32 wells at a time).
I believe at least one of my papers that compared sol and pb aCD3 had the full details on this.

lxbing0902's picture
Thank Samm for your info. Can

Thank Samm for your info. Can you explain more in detail how to calculate the amount of anti-CD3 ab needed for coating. If I use a final concentration of 2 ug/mL anti-CD3 for stimulation with a total volume of 2mL medium in a well of a 6-well plate, should I use 4 ug of anti-CD3 to prepare 1mL of PBS solution and then add this solution to the plate for coating? In this case the concentration of PBS solution is 4 ug/mL. Or should I just use 2 ug of anti-CD3 to prepare 1mL of PBS solution for coating? Thank you.

samm's picture
Hi! For plate bound Abs, your

Hi! For plate bound Abs, your coating conc is your 'final' concentration (accepted policy, even though not all your Ab may bind). Thus, for 2 ug/ml in a 6 well plate, I'd recommend 2 ug aCD3 in 1 ml of buffer (see previous post), o/n coat at ~8dC (refrigerator). Your culture will probably be done in ~2.5 ml. Before you add cells, ensure that you've added 1 ml of medium alone to 'block' the rest of the PS surface.

mforrester's picture
I have been using Samm's

I have been using Samm's suggested protocol for stimulating my T cells and it has worked really well, thank you.  Perhaps too well on ocassion (the cells have died on occassion from over stimulation) but after reducing the concentration 10-fold it has been working perfectly, thanks.

samm's picture
You raise a very good point -

You raise a very good point - I always get my techs or undergrad to do a titration when they first do these assays, since buffer, # cells, plate surface, flat v/s round bottom are all variables that need to be addressed. I've found that a range of 0.5-2 ug/ml suffices for a strong signal (some of my work deals with activation signal strengths) in 96/48 well plates, but even 0.1 ug/ml can activate cells. Also, round bottom 96 well plates seem to work the best - I even go to the extent of using 12 wells when I need a million+ cells rather than use a larger culture plate. This might be because of CD86/CD80 expression on T cells in the first few hours post activation (T:T costim).
Anyways, bottom line: 1 ug/ml is a good place to start, but once you know you can activate your cells, an additional expt to titrate out saturating conc for your culture situation is a VERY good idea.

mforrester, thanks for reminding me about this important point - this is a perfect example of 'things that don't make it into the protocol'.

NYC's picture
Hi Samm,

Hi Samm,

Quick question - when you plate with anti CD3 at 1ug/mL at 100uL/ well, do you also add the anti CD28 at .5 ug/mL at 100 uL/well?

So you have 200 uL/well incubating overnight?

Or is better to add the anti CD28 (or whatever other costimulatory molecule you're interested in) at the time of adding cells?


samm's picture
Anti-CD28 (esp the mouse 37

Anti-CD28 (esp the mouse 37.58) is a fabulous signaling Ab - so you can add it just before you add cells. My final volumes range are usually 150 ul - you want to ensure there is enough surface area:volume for gas exchange in the well, and enough volume to prevent undue evaporation (also ensure incubator humidity is high).
Adding order:
RPMI1640+5%FBS+L-glut/HEPES mod/pen-strep: qs
Anti-CD28/ICOS or IL-2 etc: add in ~10-20 ul volume
~100,000 cells: add in 50 ul volume.
Remember to precoat the aCD3 (or uncoated control wells), and wash it off. Add the RPMI first, without letting the wells dry - 15 mins with RPMI alone forms a nice 'block' too.

sandy xia
sandy xia's picture
samm, I don't know the

samm, I don't know the difference of sol and pb aCD3. How can I get your paper about this? I did search on pubmed, and got no result. For stimulation of Tcell, if I use pb aCD3, do I have to add aCD28?

samm's picture
See PMID: 16624934 in PubMed.

See PMID: 16624934 in PubMed.
You can stimulate T cells with only pb aCD3 (a small amount of costim is provided by T:T interactions), but cell proliferation is much better with aCD28.

biolin's picture
Hi Samm,

Hi Samm,
                  My work is about the activation of T cell,and I want to use anti-CD3 and anti-CD28 .
                  But I do not know which method is better,the plat bound anti-CD3 and soluble anti-CD28?
or using the anti-CD3 and anti-CD28 beads? and why? Thanks!

eringordon's picture
Does anyone know it you need

Does anyone know it you need any special kind of plate for the anti-CD3 to bind?  Can I use regular polystyrene cell culture plates.

Victor Tkachev
Victor Tkachev's picture

I'm going to stimulate murine T cells with pb anti-CD3 Abs and soluble anti-CD28 Abs. 
So, can I use BioLegend anti-CD3 Abs clone 17A2 for stimulation? If I'll use round-bottom plates and cultivate cells in final volume 100 uL, can I sorb the Abs in volume of 50 uL?


basir2020's picture
Hi Samm!!

Hi Samm!!

I planning to stimulate human PBMC for six days with anti-Cd3 and my drugs, in order to study the immunosuppressive efficacy of my drug. I am planning to culture 1ml of complete medium which will have 1 million cells. To prepare plate bound anti-CD3 for the above experiment, till what height i should coat the anti-cd3? Since i am going to culture cells in 1 ml medium, should i coat anti-cd3 till 1ml height of the plate.?

zambir's picture
 Very useful info in this

 Very useful info in this topic!

I have two questions:

(1) How long can an Ab-coated plate be preserved for?

(2) If I coat a 6-well plate but I intend to use 2 wells per day for 3 consecutive days, is it ok if I put medium in the unused wells and leave it in the incubator, until it's time to use it?


cellbiol's picture
Does anyone know any company

Does anyone know any company which produces supernatant anti-CD3 to stimulate PBLs?

Anication's picture
Hi everybody, i have a

Hi everybody, i have a problem with my Assay too, would be great, if someone has a useful tipp for me! I isolate CD4+CD62L+ t cells from mouse spleen and lymphnodes with MACS Kit.
I am coating a 96well plate with 30µl 2µg/ml aCD3e Antibody (eBioscience, functional grade, Clone 2C11) over night for 4°C.In the following procedure I am adding 1µg/ml aCD28 (eBioscience functional grade, syrian hamster) to my cells (in RPMI Medium inkl. beta-ME) and i am adding 200µl with 2x10^5 cells per Well to the coatet plate. Then I am keeping them in the Ikubator for 3 days. After these 3 days the cells are being transfered to an uncoated plate for a resting phase for 2 days. After that, the cells are being transfered to a new aCD3 coatet plate and are restimulated for another 3 days. I am taking the supernatants at every step and i want to do ELISA for produced IFNg. But it seems like my cells are not getting stimulated... they are not looking stimulated under the mikroskope and theres no IFNg... I dont know what to change and it would be very great if someone has an idea! Should i maybe use another CD3 Antibody? Thanks in advance!

SENA NUR's picture
Same problem

Hello Anika, I coated the plate and placed the cells as you did but I didnt transfer them to a new plate. I never see any proliferation, any difference. How did you overcome the problem?

th3o6a1d's picture
 Hi Samm,

 Hi Samm,

Was just wondering what the purpose of the RPMI block is.  I activated some T cells with anti-CD3, -CD28 the other day and had a really hard time getting them off the plate.  Have you ever had this happen to you?  Would the block help with this?  We used a concentration of 5 ug/mL for the anti-CD3 and 1 ug/mL for the -CD28, with 300 uL in each well of a 24 well plate.  Then, we washed twice with PBS and plated the cells.  Today, we're using a concentration of 1 and 1.