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Page 4 of 19 Mondal et al. Neuroimmunol Neuroinflammation 2018;5:34 I http://dx.doi.org/10.20517/2347-8659.2018.13

Isolation of T11TS
The glycopeptide T11TS/SLFA-3 was isolated from sheep red blood cell (SRBC) membrane. Briefly, SRBC
was trypsinized, and after nonspecific protein precipitation by TCA, was subjected to ion exchange
chromatography on a DEAE cellulose column, with a five-chambered gradient system. Finally, elute fraction
III was selected as the fraction of choice.

Administration of T11TS in animals
The first dose of 1 mL of T11TS was administered in rats intraperioneally (i.p.) from the third elute fraction
(EF III), which was followed by a second booster dose on the sixth day and the third booster dose on the day 12,
making a dose schedule of 1, 2 and 3 mL to the ET1, ET2 and ET3 animals, respectively [36,45] .

Isolation of HSC from bone marrow
From the long bones (femur, tibia, and fibula) bone marrow was isolated as described previously [41,46] . A
single cell suspension in an aseptic condition was prepared from the bone marrow. The cells were subjected
to centrifugation at 1000 rpm for 20 min on a bi-layered Percoll density gradient namely 1.077 at the bottom
and 1.050 at the top. Cells obtained from the top [i.e. low-density compartment (LDC)] and bottom [i.e.
high-density compartment (HDC)] layers were collected separately and washed thrice in PBS followed by
culturing in RPMI media as described and characterized by Chatterjee et al. , 2010.
[46]

Short-term stem cell culture
The LDC and HDC cells were cultured and maintained for 5 days in a 75 mm culture dish (Corning, USA)
containing 4 mL of RPMI-1640 supplemented with 10% FBS, 100 ng/mL of SCF, 20 ng/mL of IL3 at 37 °C in
an atmosphere of 5% CO as described by Mondal et al. , 2018.
[41]
2
Effect of T11TS on the phenotypic markers of HSCs
Bone marrow-derived isolated LDC and HDC cells of all groups were taken from 5-day cultures on the 6th
day and were subjected to flowcytometric analysis. Percentage of extracellular CD34, Sca-1 and c-kit, Ang-1
and Tie-2 population in LDC and HDC cells were evaluated using primary antibodies against CD34 (BD
Biosciences, USA), Sca-1 (Abcam Inc., USA), c-kit, Ang-1 and Tie-2 (Santa Cruz Biotechnology Inc, USA)
respectively followed by PE-conjugated anti-rat respective monoclonal antibodies for 30 min as described
by Mondal et al. , 2018 with little modification. FACS Calibur (BD Biosciences) with Cell Quest Pro
[41]
software was used for acquisition and analysis of percent cellular expression of each protein as quantified.
A total of 10,000 events were acquired and analyzed for each sample. Gating was performed with respect
to the individual group of the unstained control sample. Values indicated in “section3” are the mean of six
individual studies with S.D. calculated oneach mean value.

Immunofluorescence imaging of cells
Short term cultured HSCS grown overnight on the poly L-lysine coated sterile cover slips at 37 °C were
fixed with 4% paraformaldehyde in PBS for 20 min. After blocking, the cells were incubated overnight with
anti-primary Ang-1, Tie-2, CD34, and c-kit antibody [1:250 dilutions in PBS with 1% bovine serum albumin
(BSA)] at 4 °C. After washing with PBS, CD34 and c-kit tagged cells were incubated with FITC secondary
antibody and anti-Ang-1 and Tie-2 tagged cells were incubated with TRITC conjugated secondary antibody
(1:500 dilutions in PBS with 1% BSA). The rest of the procedures such as staining of the nucleus with DAPI,
visualization, capturing of images and quantification and representation of data were done as described [41,47] .
Briefly, Nis-Elements D3.00 were used for capturing images and quantification. Each condition was observed
in triplicate and six images were taken for each sample. Figures are representative of the group. Results were
expressed as mean fluorescence intensity of number total cells positively stained by the desired fluorescence
tagged protein out of the total number of cells counted for each experimental group. Figures are representative
of the group. As a negative control, primary antibody was omitted.