From: Syed, Farhan A. (Syed.Farhan@MAYO.EDU)
Date: Sun Feb 23 2003 - 15:00:42 EST
Dear Flowers, I am posting the answers I received on my query "Of mice and men" which i had posted sometime back. I apologize for the delay in sending the same out... I thank all of you who reponded . This forum is a great help for beginners like myself who are sometimes overwhelmed even with the simplest of issues.The comments , criticisms and insights which I got were a great source of learning! I for one have gained a lot from reading all the emails back and forth... also all the light hearted ones!! The responses I got are posted below: I have another poser today which I list at the end of all the responses I got. 1.Someone please correct me if I'm wrong, but I thought the choice of serum/Ig to block depended on the host the staining Abs were raised in. That is, if you're staining with mouse Abs, block with mouse serum/Ig. In the past I have heard someone make the case for using serum/Ig species matched to the cells to be stained instead. In this scenario human derived cells would be blocked with human serum/Ig. The logic in this case is that human Fc receptors will be more efficiently blocked by human Ig. Comments? 2., I don't see how conjugating Abs with Molecular Probes Zenon kits preclude a blocking step. Is it that you attribute all your nonspecific background to your secondary and that using the Zenon kit eliminates the need of a secondary? I would guess that you will still have a problem with a directly conjugated primary if only to a lesser extent. 3.We block with goat Ig from Sigma--much cheaper than mouse Ig. Also we are trying Molecular Probes' Zenon Abs to see if we can eliminate bloxking altogether. Beverly Barton UMDNJ-NJMS Newark, NJ USA >4. I just took a look at the immunophenotyping section of Current Protocols > in Cytometry and it says the following: > > > > 5."Intact antibodies, especially monoclonal antibodies, bind to any > cell that has unoccupied Fc > receptors. Even when the cells are derived from blood that contains all > immunoglobulin isotypes, these receptors are not fully saturated because > the immunoglobulin concentration in blood is nonsaturating. For this > reason, serum should never be used for > blocking in these protocols. It is > necessary to add a higher concentration of IgG to block all receptors so > that the antibody binds only to its epitope and not to > Fc receptors as well. All > hematopoietic cells have Fc receptors > except T cells and erythrocytes" > > > > So basically, 10% mouse serum probably wouldn't be enough to block those > pesky little Fc receptors that might be present. > > > > Their basic protocol suggest a final concentration of 200ug/ml normal > mouse IgG for blocking using a 3mg/ml stock. > > > > Probably plenty of places that you can buy it, but I took a look and > Sigma-Aldrich sells lyophilized Mouse IgG purified from serum (product# > I5831 or I8765 depending on purity) > >Hope this helps, > > 5.Huh? If human serum (and I assume mouse serum is similar) has an IgG concentration of 10 g/L (10 mg/ml), 10% serum would have a concentration of 1 mg/ml of IgG, which is 5 times more than the 200 mcg/ml you mention. > 6.Only Fc?RI and FceRI are said to be high affinity receptors. All the other Fc receptors bind antibody only complexed with antigen. How come intact antibodies, especially monoclonal antibodies, bind to any cell that has unoccupied Fc receptors? Here's my poser for the day:- This is in continuation of "The Mice and Men" question in a way.... I have been having trouble with PE labelled isotypes (IgG Kappa raised against KLH from BD and MOPC31c from sigma) which I use to define background staining. Since my epitope of interest (On surface of cells) is present on 2-3% cells of a gated population of post ficol murine marrow cells (viability by trypan blue greater than 95%) . When I stain with the isotypes stated above I see a 1-2% stain which extends right upto the top of the scale of positivity. (making it impossible to play around with the quadrant/ region) I have come up with another Idea which I want the groups "blessings " for ( I frankly do not know if someone has already tried it): The main idea behind using istype controls is if I am right , to account for non specific stain. so here's what I did and propose to use to sort my cells of interest: STEP A- Ficol prep cells (murine marrow cells) STEP B- Block with 10% Normal Mouse Serum (Todd, I checked the concn its within your suggested range) to block Fc receptors / wash STEPC - add unlabelled Isotype , incubate .wash. STEP D- add PE labelled monoclonal, incubate wash , SORT. The idea is that with STEP B, I will be accounting for stickiness due to Fc receptors and with STEP C, I will account for non Fc receptor non-specific stain(If I may call it so); since the isotype used in C is not "labelled" it will not show up as an event. I titrated the unlabelled isotype 0.1 -1ug with a constant monoclonal PE concn and found that the %+ cells goes down from 2.85 to 2.40 % a 1ugof isotype also gives back a 2.40% stained cell no. (I realize these numbers do not make sense if we see the miniscule differnce but I am dealing with low percentages to begin with) so I figured a 0.5ug Isotype concn is good enough. Downstream of the sort I will isolate RNA and check for gerne expression so as to confirm that I indded sort the cells I want. So my basic argument is that the "unlabelled" isotype will serve the purpose it is supposed to serve and at the same time not get "evented" as a false positive. Further since the isotype is in the same tube I feel it is more relevent (I know I am raising many eyebrows here) . Please let me know what you think about the approach . You are welcome to "punch" me as much as you like it really helps to clear ones thinking!!! Regards Farhan Asad Syed, Post-Doc Endocrine research Unit , Mayo Clinic. email: syed.farhan@mayo.edu > >
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