(A) Lines within the HIV vesicle in XY and YZ utilized to calculate the line profiles of most 3 stains

(A) Lines within the HIV vesicle in XY and YZ utilized to calculate the line profiles of most 3 stains. confirm co-localization of transferrin sign with p24 staining BIX02188 in both YZ and XY optical planes.(TIF) ppat.1005285.s002.tif (227K) GUID:?F3F1BBF3-6FA4-42D2-9765-A9EEF55AE276 S3 Fig: PEIC could be displaced from individual FDCs using sCD21-Ig. (A) Individual FDCs were packed with PEIC using Raji B cells in vitro, after that FDCs had been either mock treated with isotype control IgG (still left -panel) or sCD21-Ig treated (best -panel) overnight and examined the following time. After sCD21-Ig treatment no PEIC+ vesicles had been detected. In comparison, PEIC+ vesicles had been determined in the isotype treated handles. (B) The mean Mouse monoclonal to RFP Tag fluorescent strength (MFI) of 16 random fields of views (FOV) at lower magnification was compared. ****p 0.0001 (unpaired Students test).(TIF) ppat.1005285.s003.tif (553K) GUID:?40FCC691-1935-4686-8DAD-01DBC44B3F76 S4 Fig: Schematic of experimental procedure. LNs were harvested and FDCs isolated, first by depletion of CD45+ B and T cells, followed by positive selection for CD35+ BIX02188 cells. Cultures were either treated with sCD21-Ig, isotype control or RNA was directly isolated. The remaining samples were washed and HIV negative CD4 T cells were added to the FDC wells and co-cultured for 5 days. Afterwards FDCs and T cells were separated and RNA isolated. HIV RNA was quantified by ddPCR. Numbers in the graph represent total virions per well. The average cell number per well for this subject was between 15k and 150k (subject #10).(TIF) ppat.1005285.s004.tif BIX02188 (112K) GUID:?2C99BA0D-27B0-42FD-B6CF-D5829EDD83A4 S5 Fig: LN cells from one subject were analyzed by flow cytometry to determine the amount of CD35 positive T cells and to assess the contamination of CD4 positive T cells during CD35 magnetic bead positive selection. (A) CD35 expression on T and B cells shows expression on 96% of B cells as expected, interestingly 1% of T cells also express CD35, although the mean fluorescent intensity levels were lower than on B cells. (B) Before BIX02188 the positive CD35 bead selection for FDCs 79% of cells were CD3 positive. In contrast, after CD35 positive selection for FDC, only 0.3% (estimate 1 cell) was CD3 BIX02188 positive. Thus, the contribution of T cells to the CD35 positively selected FDC cultures, is negligible based on immune staining and flow cytometry.(TIF) ppat.1005285.s005.tif (127K) GUID:?E44243F1-E2A0-436F-8A25-399CF73B69AE S6 Fig: In order to determine if magnetic bead sorted-FDC in our historical samples were contaminated with CD4 T cells, we quantified CD4 mRNA levels in our FDC samples. To correlate mRNA levels with cellular contamination, we prepared a standard curve by sorting B and T cells separately and then spiking the B cells with known numbers of T cells (0 to 106 T cells in 10-fold increments, 7 data points). RNA was extracted from the B cell samples bearing a known number of contaminating CD4 T cells and the amount of CD4 mRNA quantitated using ddPCR and a standard curve prepared. (A) Standard curve of CD4 mRNA versus CD4 T cell number. (B) Extrapolation of the number of CD4 T cells in samples based on CD4 mRNA levels. Extrapolation of the number of CD4 T cells in a pure CD4 sample correlates with the number of T cells analyzed (approximately 105 cells). CD4 mRNA levels in the FDC samples indicate limited CD4 T cell contamination of the historical FDC samples. ***p 0.001 (unpaired Students test). n = 6 subjects. (C) Lymphocytes (105) from the LN of one HIV+ subject were FACS sorted and RNA was isolated as described. Then ddPCR was performed in order to determine the.