0 C. Prior to use whichever type were thawed Walls and at 10,000 g for 15 min centrifuged to remove more debris . Beadlyte M Nozzles 21 32 complexes and complex systems are used Milliplex mouse detection Rapamycin Mtor inhibitor of cytokines according to the manufacturer’s instructions to cytokines and chemokines that identify have changed after infection VER. The plates were read based on the Luminex 200 analyzer. The data were analyzed by converting mean fluorescence Th of each cytokine concentrations in picograms per milliliter with the software Beadview. For detection of TGF 1 ELISA kit was used Duoset provided by the manufacturer’s protocol. The absorbances were measured on a microplate Leseger t. Two separate tests with five M Mice per group were performed for each period.
Flow cytometry The Ph genotypes Of cells in the lung were analyzed by flow cytometry in WT Mice and Knockout St Strains determined in the country F and 24 and 48 h after infection PS-341 Proteasome inhibitor with A66. The Mice were bet Exerts and get tet, After which their lungs were immediately perfused with 10 ml of PBS and cut out. Excised lungs were then rinsed in PBS, minced with razor blades in Bo Petri dishes containing 10 ml digestion buffer and 30 g / ml DNase I in RPMI 1640 medium and placed in a incubator 37 C for 30 min. After incubation, tissues to 70-mesh sieves m were crushed and centrifuged at 1200 rpm for 7, the supernatant was removed and the cells were lysed in 5 ml of the 0th 17 M NH 4 Cl buffer and centrifuged min for 7 min. The cells were pelleted and washed in PBS, centrifuged again and the pellets were resuspended in F Rbepuffer resuspended, 1% FBS in PBS.
The cells were resuspended in an H Hlt mozytometer using trypan blue to exclude dead cells En gez. For each sample, up to 106 cells were resuspended in Lacosamide 100 l F Resuspended dyeing rbepuffer F. Rst cellular Ren FcRs with CD16/32 were for 15 min, after which the cells found with a combination of the following Abs were blocked rbt: CD3 Alexa Fluor 647 to T cells and NKT cells to identify CD19 PE determine B identify cells, FITC-Ly6G to neutrophils, CD4 PE Cy5 identify CD4 +, CD8 Alexa Fluor 750 to identify CD8 +, CD11c PEWeberCy5. 5 to dendritic cells, macrophages and F4/80 allophycocyanin identify DX5 biotin identify to identify NK and NKT cells, followed by streptavidin PECy7. The cells were incubated with Ab for 15 minutes, after which the samples were washed and resuspended in F Rbepuffer.
DAPI was immediately before the samples were collected on a LSRII added. Data were double by FlowJo foreigners Measurement of living cells in forward Backscattering and c Tea and analyzed DAPI negative. Positive Bev Lkerungen both markers were subsequently End closed. Two separate experiments were performed with three Mice in each group carried out for each period. Lungs were intracellularly Re F Staining of WT-M Mice and St Strains of knockout Mice 48 h after infection with A66 produced in the same way as for flow cytometry. A total of 106 cells were resuspended in 1 ml of 10% FBS RPMI 1640 medium in the presence of leukocyte activation mixture with GolgiPlug of 24 tissue culture plate wells and incubated at 37 C for 4 h, the cells were according to the protocol for the BD Pharmingen intracellular re F recommended staining found rbt. Briefly, cellular Re FcRs with CD16/32 for 15 min and surface Chenmarker blocked TCR FITC, Alexa Fluor 647 CD3, CD