Data confirming murine erythrocyte opsonization and oxidative damage and live microscopic analysis of oxidatively damaged erythrocyte uptake by mast cells

The data in the present article are related to research article (doi: https://doi.org/10.1016/j.imlet.2018.04.002) [1]. The data describes the detailed immunization protocol for generating polyclonal antisera to murine erythrocytes in rat. The rat anti-mouse erythrocyte serum is then tested for its ability to bind and opsonize murine erythrocytes. Second set of data confirms the oxidative damage to murine erythrocytes by treatment with different dose of the tert-butyl hydroperoxide (t-BHP) on the basis of phosphotidylserine externalization by murine erythrocytes as well as measurement of reactive oxygen species (ROS) formation in t-BHP treated erythrocytes. Third set of data depicts lack of mast cell degranulation in the form of β- hexosaminidase release in response to co-incubation of mast cell with normal and oxidatively damaged erythrocytes. Lastly, the uptake of oxidatively damaged erythrocytes by resting and activated RBL-2H3 mast cells is shown by live cell imaging using confocal microscope.


a b s t r a c t
The data in the present article are related to research article (doi: https://doi.org/10.1016/j.imlet.2018.04.002) [1]. The data describes the detailed immunization protocol for generating polyclonal antisera to murine erythrocytes in rat. The rat antimouse erythrocyte serum is then tested for its ability to bind and opsonize murine erythrocytes. Second set of data confirms the oxidative damage to murine erythrocytes by treatment with different dose of the tert-butyl hydroperoxide (t-BHP) on the basis of phosphotidylserine externalization by murine erythrocytes as well as measurement of reactive oxygen species (ROS) formation in t-BHP treated erythrocytes. Third set of data depicts lack of mast cell degranulation in the form of βhexosaminidase release in response to co-incubation of mast cell with normal and oxidatively damaged erythrocytes. Lastly, the uptake of oxidatively damaged erythrocytes by resting and activated RBL-2H3 mast cells is shown by live cell imaging using confocal microscope. &

Value of the data
This data describes the detailed immunization protocol and confirms generation of rat anti-mouse RBC serum, which can be a valuable tool for immunological research involving opsonized RBCs.
The present data could be helpful for further studies with t-BHP induced oxidatively damaged erythrocytes.
This data explains cellular and morphological changes in mast cells with time in resting and activated states and their interaction with normal or oxidatively damaged erythrocytes.
The present data provides a platform to further explore the detailed underlying mechanism of phagocytosis of erythrocytes by mast cells.

Data
The first data set shows detailed protocol to generate anti-mouse RBC serum in Wistar rat ( Fig. 1(A)). The antibody titer in rat serum was checked by hemagglutination test and found to be 1:64 ( Fig. 1(B)). The opsonization of murine erythrocytes with rat anti-mouse RBC serum (at 1:64 dilution) was confirmed using anti-rat IgG antibody by flow cytometry (Fig. 1(C)). Data showing the optimized dose of t-BHP, an oxidative stress inducing agent [2,3], based on PS externalization (Fig. 2), and the level of ROS formation by normal and t-BHP induced oxidatively damaged erythrocytes (Fig. 3) are shown.
Mast cell secretion of βhexosaminidase due to direct interaction with normal and oxidatively damaged erythrocytes is shown in Fig. 4. RBL mast cells were treated with anti DNP-IgE and allergen (DNP-BSA) for FcεRI receptor crosslinking (XL) [4] as a control. Live cell fluorescence microscopy data was collected by incubating erythrocytes with resting or activated RBL-2H3 mast cells, at 37°C. Images of resting mast cells co-incubated with oxidatively damaged erythrocytes were captured every minute for 90 min and compiled as video (movie M1). Images of activated mast cells were captured every 5 s for 60 min and compiled as video, when incubated with normal (movie M2) and oxidatively damaged erythrocytes (movie M3). Representative snapshots of uptake of oxidatively damaged erythrocytes by resting mast cells (Fig. 5), and normal ( Fig. 6) and oxidatively damaged erythrocytes (Fig. 7) Fig. 1(A). Serum was isolated and titer checked by hemagglutination test by serial dilution using 0.5% of RBC aliquots per well. Dilution of 1:64 was obtained as optimum titer  for anti-mouse RBC sera. 100 million mouse erythrocytes were incubated with rat anti-mouse erythrocyte sera at 1:64 dilution, 37°C for 30 min in continuous rotation of 20 RPM in Hybridization incubator shaker (Amerex instruments, Inc. model HS-111). Opsonization was confirmed using FITC conjugated anti-rat IgG antibody (0.2 mg) and analysis on BD FACSCalibur flow cytometer using CellQuest Pro software (BD Biosciences).

Annexin V staining
Murine (C57BL/6) erythrocytes were pretreated in vitro with tert-butyl hydroperoxide (t-BHP, 3 mM) (Sigma-Aldrich, MO, USA) at 37°C for 60 min in PBS [2]. Erythrocytes were stained with APC conjugated Annexin V (5 ng) (#640920, Biolegend, San Diego, CA, USA), washed with annexin binding buffer and analyzed on BD FACSCalibur flow cytometer as described earlier [5].   4. Mast cell secretion with normal, t-BHP treated erythrocytes and FcεRI receptor crosslinking. Percent secretion of βhexosaminidase by mast cell alone, co-incubated with normal, t-BHP treated oxidatively damaged erythrocytes and IgE crosslinked mast cells was analyzed as described in material and methods. Bar graph was plotted on the basis of βhexosaminidase secretion by mast cell in the presence or absence of normal or t-BHP treated erythrocytes and compared with secretion from IgE crosslinked activated mast cells (***P r 0.001, n ¼ 3).

Secretion assay of RBL-2H3
RBL-2H3 cells were sensitized with anti DNP IgE (TIB-142 sup), activated with DNP-BSA, and percent secretion was calculated as the percentage of total β-hexosaminidase activity released in the supernatant as described earlier [6].

Live cell imaging
0.2 Million RBL-2H3 cells were cultured overnight in RBL complete medium [1] on 35 mm petridish containing live cell imaging culture chamber (in vitro Scientific, USA). After 18 h, CFSE labeled normal or oxidatively damaged erythrocytes were co-incubated with resting or activated mast cells, in 5% CO 2 supply for 90 and 60 min respectively. Images were captured continuously at specific time lapse with Andor Spinning Disk Confocal microscope (Nikon Eclipse TiE, Software-Andor iQ 2.7) and analyzed on NIS-Element software.  6. Live cell imaging of activated mast cells co-incubated with normal eythrocytes. RBL mast cells were cultured on a glass coverslip and sensitized with IgE after 4 h. After 18 h of seeding CFSE labeled normal erythrocytes (Green) were coincubated with activated mast cells (simultaneously induced by FcεRI receptor crosslinking) at 37°C and 5% humified CO 2 (MC to RBC ratio 1:250). Images were taken every 5 s for upto 60 min continuously under fluorescence microscope and representative images from specific time point were compiled and merged. (magnification 100 Â ; scale bar, 10 mm).

Statistics
Results from at least three independent experiments are represented as mean 7 SEM. Student's t-test using Microsoft PowerPoint 2007 was performed, and a p value of less than 0.05 was considered as statistically significant. Fig. 7. Live cell imaging of activated mast cells co-incubated with oxidatively damaged eythrocytes. RBL mast cells were cultured on a glass coverslip and sensitized with IgE after 4 h. After 18 hours of seeding CFSE labeled oxidatively damaged erythrocytes (Green) were co-incubated with activated mast cells (simultaneously induced by FcεRI receptor cross-linking) at 37°C and 5% humified CO 2 (MC to RBC ratio 1:250). Images were taken every 5 s for upto 60 min continuously under fluorescence microscope and representative images from specific time point were compiled and merged. Arrow heads indicates mast cell-erythrocyte interaction; Arrows indicating uptake of erythrocytes by mast cell and circle is highlighting/showing engulfed erythrocytes inside mast cells (magnification 100 Â ; scale bar, 10 mm).