Fitc conjugated anti igmor anti igg f ab 2 secondary antibodies

We used flow cytometry to assess the reactivity of cell culture supernatants and
purified IgG from 300 patients to apoptotic Jurkat cells. Jurkat cells were exposed to UV
light (240×10⁻³ J) to induce apoptosis using a UV stratalinker
2400 (Stratagene, Santa Clara, CA). Then 1×10⁶ apoptotic Jurkat cells
were incubated for 30 minutes at 37°C with 80μl cell culture supernatants or
60μl purified IgG samples further diluted 1:2 in PBS (final dilution 1:20). After
two washes in 3ml PBS at 4°C, samples were incubated with FITC-conjugated anti-IgM
or anti-IgG F(ab’)2 secondary antibodies, respectively (Invitrogen, Camarillo, CA)
for 30 minutes at 4°C. After two additional washes in 3ml PBS at 4°C, cells
were acquired using an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA) after
gating on apoptotic cells. To avoid inter-experiment variations, all samples were assessed
at the same time in the same experiment using the same instrument settings. Results are
reported as log₂ values of the mean fluorescence intensity (MFI) of positive
cells.

The reactivity of monoclonal antibodies secreted by immortalized B-cell clones to apoptotic cells was assessed by flow cytometry, as described elsewhere.^{12 (link)} In brief, human Jurkat T leukemia cells were exposed to ultraviolet (UV) light (240 × 10⁻³ J) to induce apoptosis using a UV crosslinker (Stratalinker 2400, Stratagene, La Jolla, CA). Apoptotic Jurkat T cells were incubated for 30 minutes at 37°C with 100 µl of IgM or IgG supernatant. After 2 washes in PBS at 4°C, samples were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-IgM or anti-IgG F(ab′)₂ secondary antibodies, respectively (Invitrogen), for 30 minutes at 4°C. After 2 additional washes in PBS at 4°C, cells were acquired by flow cytometry (FACSCanto, BD Biosciences, San Jose, CA) after gating on apoptotic cells. FLOWJO software (FloJo LLC, Ashland, OR) was used to analyze the data.