For cell sorting by flow cytometry, whole blood or bone marrow from two uninfected animals were first lysed using NH4Cl, then, FcRs were blocked using cynomolgus macaque serum. Cells were counted and incubated for 30 min with the following antibodies: CD11b (ICRF44), CD45 (D058-1283), CDw125 (REA705), CD123 (7G3), CD3 (REA994), CD20 (LT20), CD8a (BW135/80), CD16 (REA423), CD10 (HI10a), CD14 (TUK4) CD32a (IV.3), and CD66 (TET2). Cell sorting was performed using a FACSAria I flow cytometer (Becton Dickinson). The sorted population was smeared with a cytocentrifuge (Cytospin 2, Thermo Scientific) and then stained using May-Grünwald Giemsa. Images were acquired using a Nikon Eclipse 80i with Dxm 1200C digital camera at 60x magnification. Cells were identified by morphological criteria by a cytologist. Promyelocytes and myelocytes were considered to be pre-neutrophils (PreN), metamyelocytes and band cells immature neutrophils, and segmented neutrophils mature cells.
Cytospin 2
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The Cytospin 2 is a centrifuge that prepares cellular samples for microscopic examination. It concentrates cells onto a slide using gentle centrifugation, allowing for the preparation of slides with a uniform cell distribution.
Automatically generated - may contain errors
4 protocols using cytospin 2
1
Flow Cytometry Cell Sorting Protocol
2
Immune Cell Profiling in Lavage Samples
3
Visualizing PBMC-Setae Interactions
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The interaction between setae and PBMC in cell cultures was observed by phase contrast microscopy. In order to visualize the interaction further, cytospin preparations followed by May-Grunwald Giemsa staining was performed on PBMCs after 24, 72 hours and 6 days of incubation with setae.
PBMCs were prepared from buffy coat as described above and 0.15×106 cells in 100 µl complete medium were added to each well of a 96-well plate. Before the cells were added to the 96-well plate 50 µl complete medium only or medium containing 400 setae was added to each well. All the cells in one well were harvested after 24, 72 hours and 6 days. The cells were washed twice in PBS containing 5% FBS and dispersed to an object glass by cytospin centrifugation according to the protocol for cytospin 2 from Shandon (Thermo Scientific). The cells were fixed in methanol for 5 minutes and stained with May-Grunwald Giemsa according to standard procedure.
PBMCs were prepared from buffy coat as described above and 0.15×106 cells in 100 µl complete medium were added to each well of a 96-well plate. Before the cells were added to the 96-well plate 50 µl complete medium only or medium containing 400 setae was added to each well. All the cells in one well were harvested after 24, 72 hours and 6 days. The cells were washed twice in PBS containing 5% FBS and dispersed to an object glass by cytospin centrifugation according to the protocol for cytospin 2 from Shandon (Thermo Scientific). The cells were fixed in methanol for 5 minutes and stained with May-Grunwald Giemsa according to standard procedure.
4
Bronchoalveolar Lavage for Leukocyte Analysis
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Immediately after the assessment of acetylcholine-induced AHR, the lungs were lavaged with 1 ml aliquots of cold Hanks' balanced salt solution (HBSS) without calcium and magnesium, containing 50 µM EDTA. The collected bronchoalveolar lavage fluid (BALF) was then centrifuged at 200×g for 10 min at 4°C. The supernatant was collected and stored at -80°C for further analysis, and the cell pellet was resuspended in cold HBSS. The number of leukocytes in the BALF was determined using a hematology analyzer (model XT-200i; Sysmex, Hyogo, Japan). Another aliquot was used for cytospin preparations at 500 rpm for 5 min (Cytospin2; Thermo Fisher Scientific, Kanagawa, Japan) and was stained with Diff-Quik for cell count (Sysmex).
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