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Microbes & Immunity Characterizing low-grade CNS tumors

(Cat. No. ab5076, Abcam, USA) with a PE-conjugated, was added to neutralize trypsin, and the suspension
anti-mouse, human cross-reactive polyclonal secondary was filtered through a 70 µm nylon mesh (HiMedia,
antibody (Cat. No. ab98742, Abcam, USA). In addition, India). The filtrate was centrifuged and resuspended in
VEGFR2 mAb (Cat. No. sc-6251, Santacruz, USA) was DMEM. Cells were counted and seeded in 60 mm culture
used with a TRITC-conjugated secondary antibody (Cat dishes (Greiner, Germany) at a density of 2 × 10 cells in
6
No. ab6786, Abcam, USA), and MMP-2 mAb (Cat. No. DMEM supplemented with 10% FBS and 2% antibiotic-
NB200-114, Novus Biologicals, USA) was paired with a antimycotic solution (HiMedia, India). Cultures were
FITC-conjugated secondary antibody (Cat. No. ab6785, maintained in a humidified incubator at 37°C with 5%
Abcam, USA). CO (New Brunswick, Eppendorf, United Kingdom) for
2
Non-conjugated primary antibodies were used at a 2 days. Following incubation, cells were detached using
1:500 dilution, while conjugated primary and secondary Accutase (Sigma-Aldrich, USA), washed, and fixed with
antibodies were used at a 1:1000 dilution. All antibodies 4% paraformaldehyde. Cell pellets were treated with
were diluted in 5% fetal bovine serum (GIBCO, USA) permeabilizing blocking buffer (5% FBS in PBS with
in PBS. Incubations were performed in a dark, humid 0.5% Tween-20) and then washed. Immunostaining was
chamber. A blocking-permeabilizing buffer was prepared performed using MMP-2 mAb (Cat. No. NB200-114, Novus
using 5% FBS in PBS containing 0.25% Tween-20 (MERCK, Biologicals, USA) at a 1:500 dilution and a PE-conjugated
India). Fluorescence imaging was conducted using the secondary antibody (Cat. No. ab97024, Abcam, USA) at
previously mentioned microscope equipped with EpiFL- a 1:700 dilution. After staining, pellets were washed and
B2A and EpiFL-G2A filters (for Alexa Fluor 488/FITC and analyzed using a BD-FACS Verse flow cytometer with
PE/TRITC, respectively; Nikon Corp., Japan). Images were Verse-Suit 1.0 software (BD Biosciences, USA).
captured and processed using the previously mentioned 2.6. Cell cycle analysis from paraffin-fixed tissue
CCD camera and imaging software. Mean fluorescence
intensity (MFI) was quantified, plotted, and subjected to Cell cycle ploidy analysis was performed on paraffin-
statistical analysis. embedded tumor tissues as previously described. 10,17
Briefly, 90 µm ribbons were sectioned from paraffinized
2.4. Immunohistochemistry with counterstaining tissue blocks and treated with xylene (MERCK, India).
Tissue sections were incubated at 54°C overnight, then The resulting pellet was collected through centrifugation,
hydrated and washed with PBS. Blocking was performed subjected to descending alcohol gradation, and washed
using 3% bovine serum albumin (LOBA, India), followed repeatedly with PBS. Antigen retrieval was performed
by overnight incubation with non-conjugated Ki-67 mAb using citrate buffer, followed by incubation. Samples
(Cat. No. sc-23900, Santacruz, USA) and DNMT1 mAb were then digested with 0.25% trypsin-EDTA, washed
(Cat. No. sc-271729, Santacruz, USA) at a 1:200 dilution. The in PBS, and filtered through a 70 µm nylon mesh. The
slides were then washed and incubated with a horseradish filtrate was centrifuged and resuspended in PBS. RNaseA
peroxidase-conjugated secondary antibody (Cat. No. (1 mg/mL; Invitrogen, USA) was added and incubated at
ab97030; Abcam, USA) at 1:500 in ×1 PBS. Development 37°C. Propidium iodide (PI; 0.5 mg/mL; Life Technologies,
was achieved using 3,3-diaminobenzidine (SRL, India) in USA) was then added, followed by incubation. Samples
buffered H O (1M TRIS, pH 7.4) supplemented with 0.5% were analyzed using the BD Accuri C5 flow cytometer (BD
2
2
cupric sulfate, in the dark for 20 minutes. Sections were Biosciences, USA). The cumulative percentage of cells in
counterstained with hematoxylin (Merck, India), followed the S+G2M phases was used as an indicator of proliferation
by graded alcohol dehydration, air drying, and mounting. and was subsequently graphed and analyzed statistically.
Slides were examined using the same microscopic system.
For Ki-67 quantification, the number of Ki-67-positive cells 2.7. Statistical analysis
were counted and divided by the total number of cells within All experiments were performed in triplicate for
each microscopical field to calculate the proliferative index. each sample. Depending on the data distribution and
Data were tabulated, graphed, and statistically analyzed. experimental design, non-parametric tests and/or t-tests
were applied. Statistical significance was assessed using
2.5. Cell isolation, culture, and immunophenotyping the Kruskal–Wallis test and nested one-way ANOVA,
®
Freshly excised tumor samples were aseptically collected performed with GraphPad Prism 10 for Windows 64-bit,
in serum-free DMEM at 4 – 6°C and mechanically version 10.4.2 (633). A p≤0.05 was considered statistically
minced. Samples were enzymatically dissociated using significant. Additional statistical tests were employed where
0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) appropriate. Full statistical details, including significance
(Sigma Aldrich, USA). FBS (MP Biomedicals, USA) levels, are provided in the figure legends.

Volume 2 Issue 3 (2025) 133 doi: 10.36922/MI025190040