Flow cytometry data were collected using a FACSCalibur instrument (BD Biosciences) equipped with red and blue lasers. A minimum of 50,000 total cells were collected for each sample. The acquired data were analyzed using FCS Express 4 Flow Cytometry (De Novo Software; version 4.07). To measure the levels of dsRNA, infected cells (MOI=10) were harvested via trypsin digestion and washed once with 1× PBS containing 1% BSA (Gemini BioProducts). Cells were then fixed and permeabilized for 20 min. using the Cytofix/Cytoperm kit (BD Biosciences). After this fixation step, cells were washed twice with 1× Perm/Wash Buffer (BD Biosciences) before adding the J2 anti-dsRNA monoclonal antibody (Scicons). After 45 min. of incubation at room temperature, the cells were washed once with 1× Perm/Wash Buffer followed by the addition of anti-mouse IgG2a APC (eBioscience) for 45 min. A final wash with 1× Perm/Wash Buffer was performed prior to the addition of 2% paraformaldehyde (Electron Microscopy Sciences).
Anti mouse igg2a apc
Manufactured by Thermo Fisher Scientific
The Anti-mouse IgG2a APC is a lab equipment product that is used for the detection and quantification of mouse IgG2a antibodies. It is a fluorescently-labeled secondary antibody that binds to the IgG2a subclass of mouse immunoglobulins. The APC (Allophycocyanin) fluorescent dye provides a bright signal for flow cytometry and other immunoassay applications.
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Lab products found in correlation
Fcs express 4 flow cytometry, by De Novo Software (1 mentions)
Perm wash buffer, by Thermo Fisher Scientific (1 mentions)
J2 anti dsrna monoclonal antibody, by Techcomp Instruments (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Bovine serum albumin (bsa), by Gemini Bio (1 mentions)
Facscalibur, by BD (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
Perm wash buffer, by BD (1 mentions)
Cell viability kit, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
2 protocols using anti mouse igg2a apc
1
Quantifying dsRNA Levels in Infected Cells
2
Complement Deposition Assay for Bacterial Quantification
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The complement deposition assay was based on a previously published method41 (link). Briefly, bacteria was added to brain heart infusion (BHI) broth, incubated at 37 °C for 15 min, and centrifuged. The supernatant was removed and the pellet was washed and re-suspended for incubation in PBS with 20% human serum (pooled from five individuals) and 1% gelatin veronal buffer. After washing, the pellets were re-suspended in mouse-anti-human-C3 in PBS (Abcam) and incubated at 37 °C for 30 min. Washing was repeated, and the contents were re-suspended in anti-Mouse IgG2a-APC in PBS (EBioscience) and incubated at 4 °C for 30 min in the absence of light. After washing, the remaining bacteria were re-suspended in PBS and incubated with thiazole orange (BD Cell Viability kit). Samples were acquired using the Accuri C6 flow cytometer (BD).
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