Follicular fluid aspirated from follicles of individuals during oocyte retrieval was pooled and considered as independent samples. Granulosa cells were isolated from the follicular fluid as previously described [26 ]. Briefly, follicular fluid was centrifugated at 1200 rpm for 10 min to precipitate cells. The cell pellets were resuspended in PBS and layered onto a 50% Percoll Reagent (Sigma-Aldrich, MO, USA) and centrifugated at 1200 rpm for 30 min to separate granulosa cells from blood cells. Granulosa cells at the interface were harvested and washed with PBS and red blood cell lysis buffer (Servicebio, Wuhan, China). After centrifugation at 1500 rpm for 15 min, granulosa cells were collected and used for RNA extraction.
Red blood cell lysis buffer
Manufactured by Wuhan Servicebio Technology
Sourced in China
Red blood cell lysis buffer is a solution used to selectively lyse (break down) red blood cells in a sample, while leaving other cell types intact. This buffer allows for the separation and isolation of non-red blood cell components, such as leukocytes, for further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Percoll reagent, by Merck Group (1 mentions)
Ab203445, by Abcam (1 mentions)
Ab197730, by Abcam (1 mentions)
15 ml falcon tube, by Corning (1 mentions)
Phosphate buffered saline (pbs), by Wuhan Servicebio Technology (1 mentions)
Pe anti mouse foxp3, by BioLegend (1 mentions)
Dnaase 1, by Merck Group (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
6 protocols using red blood cell lysis buffer
1
Granulosa Cell Isolation from Follicular Fluid
2
Whole Blood Leukocyte Isolation and Immunofluorescence
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We transferred 4 ml of healthy donors’ whole blood to a 15-ml Falcon tube (Corning, Lowell, MA, USA) and gently mixed it with red blood cell lysis buffer (Servicebio, Wuhan, China). After approximately 5 min (when the color of the blood changed to a transparent cherry red), cells were immediately centrifuged at 400 g for 10 min at 4 °C. They were resuspended in PBS and coated on polylysine-coated slips, then fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% Triton X-100 (10 min), blocked with 2% BSA (30 min), and stained with 30 μg/mL of DAPI, Alexa Fluor®488 conjugated anti-CD45 antibody (ab197730, Abcam, Cambridge, UK), and Alexa Fluor®568 conjugated anti-Cytokeratin 19 antibody (ab203445, Abcam, Cambridge, UK). After thoroughly washing with PBST, the cells were coverslipped using antifade mounting medium. As a negative control, the primary antibody was omitted, and all of the other steps were similar to those described. Fluorescence images were obtained by Olympus fluorescence microscope BX63 (Olympus, Japan).
3
Inhibiting Lactate Excretion in Tumor Microenvironment
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C57BL/6J mice were subcutaneously injected 150 µL GL261 cells (4 × 106) containing with 100 µm of sodium bicarbonate (SB) or 2 nm of BAY‐876. After 5 days post‐injection of tumor cells, the tumor growth was starting to be monitored, and the tumor volume was calculated according to the formula V = L*W*W/2 (L represented the longest dimension, and W represented the shortest dimension).
To measure the content of LA, TNF‐α, and IFN‐γ in TME, the tumor tissues were homogenized using a homogenizer. After centrifugation at 12 000 rpm for 10 min, the LA concentration was measured using LA assay kits, and the TNF‐α and IFN‐γ concentrations were measured by mouse TNF‐α and IFN‐γ ELISA kits according to the manufacturer's instructions.
To reveal the ITM‐alleviation mechanism of lactate‐excretion inhibition, the tumor tissues were dissociated into cell suspension for FCM examination. After treatment with red blood cell lysis buffer (Servicebio, G2015, China), the cells were stained with anti‐CD45‐PE antibody, anti‐CD11b‐PerCP‐Cy5.5 antibody, and anti‐CD206‐APC antibody for M2Φ analysis; anti‐CD3‐PerCP‐Cy5.5 antibody anti‐CD4‐FITC antibody, anti‐Foxp3‐APC antibody for Tregs.
To measure the content of LA, TNF‐α, and IFN‐γ in TME, the tumor tissues were homogenized using a homogenizer. After centrifugation at 12 000 rpm for 10 min, the LA concentration was measured using LA assay kits, and the TNF‐α and IFN‐γ concentrations were measured by mouse TNF‐α and IFN‐γ ELISA kits according to the manufacturer's instructions.
To reveal the ITM‐alleviation mechanism of lactate‐excretion inhibition, the tumor tissues were dissociated into cell suspension for FCM examination. After treatment with red blood cell lysis buffer (Servicebio, G2015, China), the cells were stained with anti‐CD45‐PE antibody, anti‐CD11b‐PerCP‐Cy5.5 antibody, and anti‐CD206‐APC antibody for M2Φ analysis; anti‐CD3‐PerCP‐Cy5.5 antibody anti‐CD4‐FITC antibody, anti‐Foxp3‐APC antibody for Tregs.
4
Isolation and Characterization of Odontogenic Keratocyst Cells
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Three OKC tissue samples were collected from the Department of Oral and Maxillofacial Head Neck Surgery, School & Hospital of Stomatology, Wuhan University. The patients agreed and signed the informed consent form, and ethical approval was acquired from the Ethics Committee of the School of Stomatology, Wuhan University (Wuhan, PR China). The processing of clinical samples was conducted in accordance with the Declaration of Helsinki. Specimens of OKC were carefully washed with Hanks solution for 5 times and cutted into 2–3 mm pieces. Then, 2 mL GEXSCOPE® Tissue Dissociation Solution was used for the tissue pieces digestion at 37°C for 15 mins in 15 mL tubes. The solutions were filtered using 40-micron sterile strainers and then centrifuged at 200 × g for 3 mins. After centrifugation, the supernatant was carefully removed, and the deposit was gently suspended in 2 mL phosphate buffered saline (PBS) (Servicebio, Wuhan, China). Red blood cells were removed using 3 mL red blood cell lysis buffer (Servicebio, Wuhan, China) which was incubated with the collected cells at 37°C for 5 mins. Then, the buffer was removed by centrifugation with speed of 200 × g for 3 mins. The pellet was suspended in 2 mL PBS and stained by trypan blue (Servicebio, Wuhan, China). The stained suspension was observed and evaluated in microscope.
5
Comprehensive Immunological Evaluation Protocol
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DNCB was purchased from Sigma (Cat. No. 237329-10G, Missouri, USA). Olive oil was purchased from Pythonbio (Guangzhou, China). HITRAP PROTEIN G HP 1×5 ML was purchased from Cytiva (Cat. No. 17040501, Shanghai, China). Foxp3 antibody was purchased from Affinity Biosciences (Cat. No. AF6544, Jiangsu, China). Red blood cell lysis buffer was purchased from Servicebio (Cat. No. G2015-500ML, Wuhan, China). Cell Activation Cocktail (with Brefeldin A) (Cat. No. 423303), FITC anti-mouse CD4 (Cat. No.130308), PE anti-mouse IFN-γ (Cat. No.505808), APC anti-mouse IL-10 (Cat. No.505009), PE anti-mouse FoxP3 (Cat. No.126404) were obtained from Biolegend (San Diego, USA). Mouse IgG (Cat. No. EMC116.96), IgE (Cat. No. EMC117.96), IL-10 (Cat. No. EMC005.96) and IFN-γ (Cat. No. EMC101g.96) enzyme-linked immunosorbent assay (ELISA) kits were all purchased from Neobioscience Biotechnology Company (Shenzhen, China). The Diaminobenzidine (DAB) chromogenic agent kit (Cat. No. G1211) was procured from Servicebio in Wuhan, China. TRIzol reagent was purchased from Thermo Scientific (Cat. No. 15596018, MA, United States). ReverTra Ace qPCR RT Kit (Cat. No. FSQ-101) and SYBR Green Master Mix (Cat. No. QPK-201C) were purchased from Toyobo (Shanghai, China).
6
Isolation of Murine Leukocyte Populations
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Preparation of single-cell suspensions of murine splenocytes was performed according to a protocol described previously. 22 Single-cell leukocyte suspensions of murine peripheral blood were prepared by lysing erythrocytes with red blood cell lysis buffer (Servicebio). Mouse liver infiltrated lymphocytes (LILs) were isolated as described previously. 20 Briefly, mouse livers were perfused with 10 mL phosphate buffer solution (PBS) immediately after sacrifice. The livers were then homogenized and digested with enzyme solution containing 0.05% collagenase type IV (Sigma-Aldrich), 0.002% DNAase I (Sigma-Aldrich), and 10% fetal bovine serum for 30 min. After digestion, the pellet was resuspended in 40% Percoll and centrifuged at 1000g without break.
After removing the debris and hepatocytes on the top layer, LILs in the pellet were collected, washed, and subjected to further analysis.
After removing the debris and hepatocytes on the top layer, LILs in the pellet were collected, washed, and subjected to further analysis.
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