Journal: Journal of Translational Medicine

Article Title: AQP5-1364A/C polymorphism and the AQP5 expression influence sepsis survival and immune cell migration: a prospective laboratory and patient study

doi: 10.1186/s12967-016-1079-2

Figure Lengend Snippet: Western blot and migration assay of transfected Jurkat-cells and AQP5-immunostaining of neutrophils obtained from healthy donors a Western blot from whole cell lysates of Jurkat-cells ( lane u untransfected cells, lane c transfected with control plasmid, lanes 1 – 3 AQP5_pReceiver transfected Jurkat cells) with AQP5 and ACTB antibody; As indicated by the stronger band in lanes 1 – 3 , Jurkat cells could successfully be stable transfected; b Migration assay performed with cell culture inserts utilizing transfected Jurkat cells stimulated with SDF-1α and counted after 22 h (n = 6, 6); AQP5 overexpressing (AQP5_pRec) cells showed increased migration (p = 0.009) compared to control cells (pRec). c Representative examples of immunostaining with AQP5 antibody of neutrophils obtained from two healthy AA genotype carriers showing a considerable amount of AQP5 protein ( brown precipitate ). In neutrophils of two CC genotype carriers no clear AQP5 protein expression is detected. Every staining was performed in duplicate and eight random photographs were taken from each slide, 40× magnification

Article Snippet: Stable transfection with Jurkat T-cells was performed using an AQP5_pReceiver EX-T1015-M09 vector (Human Full ORF-Clone, NM-001651, pReceiver-M09) or a pReceiverM09 (GeneCopeia, Rockville, MD, USA).

Techniques: Western Blot, Migration, Transfection, Immunostaining, Control, Plasmid Preparation, Cell Culture, Expressing, Staining

Journal: Blood

Article Title: Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin

doi: 10.1182/blood-2009-09-244251

Figure Lengend Snippet: Multiple CLL mAbs react with MEACs. Spontaneous apoptotic Jurkat cells were stained with rabbit anti–human MYHIIA antibody (25 μg/mL) and CLL mAb (25 μg/mL), followed by secondary antibodies: PE-conjugated anti–rabbit IgG (1 μg/mL) and FITC-conjugated anti–human IgG (1 μg/mL). (A) Flow cytometric analyses are displayed as contour plots of fluorescence intensity shown on log scales with BiExponential transformation. Top left panel shows cells stained with secondary antibodies alone. Top middle panel shows cells stained with all reagents except CLL mAb. The remaining panels show staining with different CLL mAbs as indicated. (B) After gating on MYHIIA+ cells, histograms of the percentage of maximum (% of Max) fluorescent intensity for indicated CLL mAb staining (blue thick line) is shown relative to cells stained with all reagents except CLL mAb (red thin line). CLL 258 histogram (not shown) was similar to that of CLL 068. (C) Chart showing the MFIR staining of MEACs with 26 CLL mAbs. The MFI for CLL mAb staining of MYHIIA+ cells was determined from histograms as shown in panel B. The MFIR was calculated by dividing with the MFI obtained for MYHIIA+ cells that were stained with all reagents except CLL mAb. The CLL mAb is indicated on the x-axis. The CLL mAb subset is indicated underneath the CLL mAb numbers (subset number or “-” if not part of a subset). The bottom row of letters indicates if the IGHV of a CLL mAb is mutated (M) or not (U).

Article Snippet: A human T-cell line (Jurkat) was cultured at 2 × 10 5 cells/mL in RPMI 1640 (Mediatech) supplemented with 10% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine (Invitrogen) at 37°C and 5% CO 2 .

Techniques: Staining, Fluorescence, Transformation Assay

Journal: Blood

Article Title: Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin

doi: 10.1182/blood-2009-09-244251

Figure Lengend Snippet: Apoptosis exposes MYHIIA and permits CLL subset 6 mAb reactivity. (A-B) Spontaneous apoptosis in Jurkat cells was revealed by propidium iodide (PI; red)–stained DNA in condensed nuclei. Apoptotic cells were costained under nonpermeabilizing conditions with (A) anti-MYHIIA antibody (green) or (B) CLL 068 mAb (green) and visualized by confocal microscopy (original magnification, ×600). Two representative cells from 6 independent experiments are shown with the average Pearson correlation coefficient (r) as a measure of colocalization. (C) Spontaneous apoptotic Jurkat cells were stained under nonpermeabilizing conditions with anti-MYHIIA (red) and CLL 068 mAb (green) and visualized by confocal microscopy (original magnification, ×600). Two representative cells from 3 independent experiments are shown with the average r. In all experiments (A-C), merges of red and green panels are shown (Merge; overlap in yellow-orange) and staining with secondary antibody alone was negative (not shown).

Article Snippet: A human T-cell line (Jurkat) was cultured at 2 × 10 5 cells/mL in RPMI 1640 (Mediatech) supplemented with 10% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine (Invitrogen) at 37°C and 5% CO 2 .

Techniques: Staining, Confocal Microscopy

Journal: Blood

Article Title: Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin

doi: 10.1182/blood-2009-09-244251

Figure Lengend Snippet: MYHIIA and CLL subset 6 mAb reactivity is exposed on a subset of early and late apoptotic cells. Flow cytometric analyses of spontaneous apoptotic Jurkat cells are displayed as contour plots of fluorescence intensity shown on 4-log scales with BiExponential transformation. (A) Cells were stained with rabbit anti–human MYHIIA antibody (100 μg/mL), FITC-conjugated anti–rabbit IgG (3 μg/mL), 7AAD, and AV-PE. Representatives of 9 experiments are shown. Early apoptotic cells (7AAD−, AV-PE+, 26.3%; top left panel) were gated and contain a subset of MYHIIA+ cells (22.9%; bottom right panel). Late apoptotic cells (7AAD+, AV-PE+, 14.5%; top left panel) were gated and contain a subset of MYHIIA+ cells (9.4%; top right panel). Gated live cells (7AAD−, AV-PE−, 58.5%; top left panel) contained no MYHIIA+ cells (0.4%; bottom left panel). (B) Same experiment as in panel A showing MYHIIA+ cells (7.3%) are all AV-PE+ (top panel). Gated MYHIIA+ cells are either early (78.9%) or late (19.9%) apoptotic cells (bottom panel). (C) Cells were stained with human CLL 068 mAb (100 μg/mL), FITC-conjugated anti–human IgG (2 μg/mL), 7AAD, and AV-PE. Representatives of 23 experiments are shown. Early apoptotic cells (23.7%; top left panel) were gated and contain a subset of CLL 068+ cells (27.3%; bottom right panel). Late apoptotic cells (23.0%; top left panel) were gated and contain a subset of CLL 068+ cells (8.3%; top right panel). Gated live cells (52.4%; top left panel) contained no CLL 068+ cells (0.1%; bottom left panel). (D) Same experiment as in panel C showing CLL 068+ cells (8.7%) are all AV-PE+ (top panel). Gated CLL 068+ cells are either early (73.6%) or late (24.8%) apoptotic cells (bottom panel). (E) Cells were stained with CLL 068 mAb (50 μg/mL) plus FITC-conjugated anti–human IgG (0.2 μg/mL; x-axis) and PE-conjugated anti–rabbit IgG alone (2.4 μg/mL), AV-PE, or anti-MYHIIA antibody (200 μg/mL) plus PE-conjugated anti–rabbit IgG (y-axis; left to right, respectively). Representatives of 19 experiments are shown. In this experiment, CLL mAb 068 reacts with 36.6% cells with less than 1% background PE staining (left panel). CLL 068 only stains AV-PE+ cells (44.1%), with a subset of AV-PE+ cells negative for CLL 068 staining (19.8%; middle panel). All CLL 068+ cells are MYHIIA+ (42.8%; right panel). Cells were not stained by secondary antibodies alone (not shown).

Article Snippet: A human T-cell line (Jurkat) was cultured at 2 × 10 5 cells/mL in RPMI 1640 (Mediatech) supplemented with 10% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine (Invitrogen) at 37°C and 5% CO 2 .

Techniques: Fluorescence, Transformation Assay, Staining

Journal: Blood

Article Title: Many chronic lymphocytic leukemia antibodies recognize apoptotic cells with exposed nonmuscle myosin heavy chain IIA: implications for patient outcome and cell of origin

doi: 10.1182/blood-2009-09-244251

Figure Lengend Snippet: Normal human serum IgG reacts with MEACs. Spontaneous apoptotic Jurkat cells were stained with 25 μg/mL rabbit anti–human MYHIIA antibody and 25 μg/mL CLL mAb, normal human serum IgG from Sigma (S; 25 μg/mL) or Miltenyi Biotec (M; 2 μL), or 25 μg/mL humanized anti-CD20 mAb, followed by 1 μg/mL secondary antibodies: PE-conjugated anti–rabbit IgG and FITC-conjugated anti–human IgG. Representatives of 4 experiments are shown. (A) Flow cytometric analyses are displayed as contour plots of fluorescence intensity shown on log scales with BiExponential transformation. Panels show costaining with anti-MYHIIA and serum IgG or anti-CD20. (B) After gating on MYHIIA+ cells, histograms of the percentage of maximum (% of Max) fluorescent intensity for indicated staining (thick blue line) is shown relative to cells stained with CLL 169 mAb (thin red line). (C) The MFIR staining of MEACs was calculated as in Figure 3C except that the MFIR was calculated by dividing the MFI for MYHIIA+ cells stained with CLL 169 mAb.

Article Snippet: A human T-cell line (Jurkat) was cultured at 2 × 10 5 cells/mL in RPMI 1640 (Mediatech) supplemented with 10% heat inactivated fetal bovine serum (FBS; Atlanta Biologicals), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine (Invitrogen) at 37°C and 5% CO 2 .

Techniques: Staining, Fluorescence, Transformation Assay