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anti-human IgG,A,M,D,E in supernatants for cytometry

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Old 02-24-2012, 12:58 PM
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Default anti-human IgG,A,M,D,E in supernatants for cytometry



Can anybody tell me which, among the many types of monoclonal antibodies, should I use in order to catch, by flow cytometry, immunoglobulins from supernatants? Whole molecules or some chains? I can work only on 3 colours- with FITC, RPE-Cy5, PE.

(Another question, for which I haven't found an answer through almost 4 years, what kind of immunoglobulins can I find in supernatants after 72h and 120h of cell culture stimulation? I bet that I should have IgM and IgG at least.)

Please, help.
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Old 03-06-2012, 04:17 PM
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Default Re: anti-human IgG,A,M,D,E in supernatants for cytometry

For Ig synthesis and secretion from B cells obligatory participants in the process are null cells, that produce human immunoglobulin synthesis/secretion-facilitating factor (HISFF), T cells, monocytes and PWM, and for to maximize production the culture must be prolonged for up to 4 days for high secretion of IgA and IgG and up to 6-7 days for high IgM/IgG secretion, replacing every 2 days half of the culture medium and each time medium subtracted is retained. The secreted antibody concentration is positively correlated with PBMC concentration and incubation time in the tissue culture and negatively correlated with storage time of the whole blood at room temperature. In addition, is consistently observed that IgM is secreted in much greater amounts by the cultured B cells than IgG, and that IgG not’s secreted in the absence of IgM.
From my work “ Zagami F.: Proliferative response of the lymphocytes to mitogens (1994/1995) Giorn. It. Patol. Clin. Vol. IX/X, N° 1-4, pag. 28 – 34, Mar.-Dic. 1994/1995” PBMC are isolated from blood samples via Histopaque layering 6 ml of blood is collected in citrate anticoagulant and diluted with sterile DPBS at up to 8 ml in a 10 ml sterile glass conical tube. The diluted blood is split into two tubes and layered onto 2 ml of Histopaque-1077 (Sigma H8889) in a sterile 10 ml conical tube without mixing, and centrifuged at 1200 rpm (290 g) for 30 min. The mononuclear cell layer is transferred to a new tube and washed with 1× PBS. The cells are centrifuged at 1200 rpm for 5 min in 8 ml of PBS. The cell pellet was resuspended in 2 ml of PBS and determine the PBMC concentration/viability with ethidium bromide/acridine orange on hemocytometer, and adjusted to a concentration of 107 cells per ml with complete RPMI 1640 medium (50 ml of 10 % fetal calf serum [FCS], 10 ml of 2 % L-glutamine, and 5 ml of antibiotics [amphotericin B-penicillin-streptomycin, 1% {Mixed}]) is added to every 500 ml of RPMI medium using a sterile technique. The medium is filtered via a 0.22 μm pore size filter if a precipitate appeared. The complete medium is stored at 4°C for up to 30 days) and the cells incubated at 37 °C in 5% CO2 for 90, 120 and up to 168 h. At the end of the incubation period, the tissue culture fluid is removed and stored at −20°C.
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Old 07-02-2012, 03:36 PM
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Default Re: anti-human IgG,A,M,D,E in supernatants for cytometry

I have just found out that someone replied. I also use Histopaque-1077. I ordered kit for Igs analysis. It should work for me. Thanks a lot for answer.
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