Accucount fluorescent particles

Microparticles were purified as previously described^{23 (link),63} . Briefly, 1 × 10⁶ HaCaT or PDV cells were seeded and incubated overnight. Supernatants were collected from confluent cells, and centrifuged at 1500 × g for 5 min to remove cells and cellular debris. The supernatants were then centrifuged at 17,000 × g for 20 min at 4 °C to pellet the microparticles.
Microparticles were quantified using flow cytometry. Purified microparticles were resuspended in annexin binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl₂) and stained with 1.25 µl annexin V Alexa Fluor 488 (Invitrogen), in order to detect phosphatidylserine, then analysed using flow cytometry. The sizing gate (0.1–1 μm) was defined using spherical beads ranging from 0.7–7.4 µm (Spherotech) that were added to the sample. In addition to size, microparticles were distinguished by the expression of phosphatidylserine on the outer surface (positive annexin V staining). The absolute number of microparticles in each sample was determined using a defined number of AccuCount fluorescent particles (Spherotech). All analysis of flow cytometry data was carried out using FlowJo vX software.

To generate effector CTL, lymph node cells from Ly5.2 F5.Rag1-/- mice were activated in vitro for three days in the presence of NP68 peptide (10⁻⁸ M). Activated blasts were purified by Ficoll (GE Healthcare) density-gradient centrifugation and expanded for a further four days in the presence of 10 nM IL-2 (Peprotech). Ly5.1+ target cells were prepared as described above. CTL and target cells were added to wells at an E:T ratio of at least 5∶1, and briefly centrifuged to initiate cell contact. Cells were co-cultured at 37°C for the indicated period of time (10 minutes – 2 hours) in the presence of anti-LAMP1a (eBioscience) to detect degranulation of CTL during the culture period. At the end of the culture period, cells were immediately fixed with IC fixation buffer (eBioscience) to preserve E:T conjugates. Samples were then stained and analysed by flow cytometry, with the addition of a known number of AccuCount fluorescent particles (Spherotech) to determine cell counts. Target and effector cells were identified by expression of Ly5.1 or Ly5.2 respectively, and E:T conjugates by dual fluorescence for these markers along with forward scatter area and width characteristics to identify doublets. Staining for TCRβ and B220 was used to identify T and B cell targets, and CTV fluorescence to identify cells that had been pulsed with different doses of peptide.

Whole blood (100 μl) was stained for Ly-6G (neutrophils, DCs) (clone 1A8, BD Bioscience), CD14 (monocytes) (clone rmC5–3, BD Bioscience), and F4–80 (monocytes) (clone BM8, Biolegend) to quantitate granulocytes. AccuCount Fluorescent Particles (12.5 μl of 5.2 μm size, Spherotech, Inc.) were added to calculate absolute counts (cells/μl). Whole blood was lysed after staining with FACS lysis buffer (BD Bioscience) and analyzed on a FACS Canto A (BD Bioscience) with DiVa Software and further analyzed using FlowJo Analysis Software (Tree Star Inc.). Cells were gated for forward and side scatter and dead cells (a very small fraction) were excluded. The total numbers of granulocytes were quantified as: (the number of events (Ly-6G high, CD14-, and F4–80-) divided by (number of events for the AccuCount particles) times (number of AccuCount particles per 12.5 μL times volume of test sample used (0.1 ml)). In some samples, absolute counts of granulocytes were measured using volumetric/flow-rate calibration [35 (link),36 (link)]. Stained and lysed whole blood was analyzed for a fixed amount of time that resulted in identical amounts of volume analyzed. All samples from each experiment were analyzed at the same time to avoid possible variation in flow rates that could occur at different days, temperatures, or relative humidities.

Whole lungs were removed and chopped with razor blades, incubated with type IV collagenase (Worthington) at 37 °C for 40 min, then homogenized through a 70-µm cell strainer (Falcon). Remaining red blood cells were lysed using 1× red blood cell lysis buffer (BD Biosciences). Cells were stained with Fixable Viability Dye eFluor^® 455 (eBioscience). Anti-mouse immunophenotyping antibodies were diluted in FACS buffer (3% FBS, 2 mM EDTA, 1× PBS) to 5 µg per mL along with Fc block (anti-mouse CD16/CD32; 5 µg per mL, BD), and cells were stained for 30 min on ice in three groups (AMφ: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), and Siglec-F (E50-2440; BD); monocyte and neutrophil: Ly-6C (AL-21; BD), Ly-6G (1A8; BD), and CD11b (M1/70; BD); dendritic cell: CD45 (30-F11; BD), CD11c (HL3; BD), CD11b (M1/70; BD), MHC II (M5/114.15.2; BD), and CD103 (M290; BD); NK cells: CD3 (17A2; BD), NK-1.1 (PK136; BD), and CD49b (DX5; BD)). After the staining, cells were washed twice with FACS buffer and then fixed in 2% para-formaldehyde in FACS buffer for 15 min. Cell numbers were counted using AccuCount Fluorescent Particles (Spherotech). All data were collected on an LSR II flow cytometer (BD) and analyzed using FlowJo software.

Whole blood (50 µl) was stained for CD14 (monocytes) (clone rmC5-3, BD Biosciences), CD11b (activation - monocytes, macrophages, granulocytes, and NK cells) (clone M1/70, BD Biosciences), F4-80 (monocytes) (clone BM8, Biolegend), and Ly-6G (neutrophils, DCs) (clone 1A8, BD Biosciences) to quantitate granulocytes. AccuCount Fluorescent Particles (12.5 µl of 5.2 µm size, Spherotech, Inc.) were added to determine absolute counts (cells/µl). Whole blood was lysed after staining with FACS lysis buffer (BD Biosciences) and analyzed on a FACSVantage (BD Biosciences). Cells were gated for forward and side scatter and dead cells (a very small fraction) were excluded. Ten thousand events (excluding beads) were obtained. Granulocytes (Ly-6G⁺, CD14⁻, F4-80⁻) were quantified. In some samples, absolute counts of granulocytes were measured using volumetric/flow-rate calibration [40] (link), [41] . Stained and lysed whole blood was analyzed for a fixed amount of time that resulted in identical amounts of volume to be analyzed. The total number of granulocytes were then calculated based on the volume of the 50 µl of blood analyzed. All samples from each experiment were analyzed at the same time to avoid possible variations in flow rates that could occur at different days, temperatures, or relative humidities.