Human plasma was divided into eight EP tubes. The plasma content of each tube was 1200 µL, and red blood cell lysate content was 6, 12, 15, 21, 30, 42, or 72 µL (Red Blood Cell Lysis Buffer, B541001; Sangon Biotech). Serially diluted hemolysis samples were prepared. The hemolysis index (HI) was calculated as follows: HI = A 414 - A 385 + 0.16 × A 385 6 (A414 and A385 reflect absorption peaks at 414 and 385 nm, respectively). Hemolysis was measured using a spectrophotometer (Youke Instrument) and hemocytometer (Coulter HL780; Beckman Coulter).
Red blood cell lysis buffer
Manufactured by Sangon
Sourced in China
Red Blood Cell Lysis Buffer is a solution used for the selective lysis of red blood cells in a biological sample. It is designed to remove erythrocytes while leaving other cell types intact, facilitating the isolation and analysis of the remaining cellular components.
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5 protocols using red blood cell lysis buffer
1
Quantification of Hemolysis in Plasma Samples
2
Blood Cell Lysis and Analysis
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Approximately 0.5 mL blood from the internal canthal vein was collected in heparin following orbital enucleation. Erythrocytes in the samples were lysed by adding 3 mL red blood cell lysis buffer (Sangon Biotech, Shanghai, China) for 5 min at room temperature (approximately 25°C). Cells were then washed once and resuspended in fluorescence activated cell sorting (FACS) buffer for flow cytometric analysis.
3
Tumor Dissociation and Immune Analysis
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Tumors were excised at the endpoint, cut into small pieces, and digested with 1 mg/mL collagenase (MilliporeSigma) for 40 minutes in a 37°C shaking incubator. Cell suspensions were filtered through a 70 μm cell strainer, and RBCs were removed using Red Blood Cell Lysis Buffer (B541001, Sangon Biotech) according to the manufacturer’s protocol. Cells were collected, and PD-L1 expression and TILs were analyzed by flow cytometry.
4
Isolation of CD11b+ Cells from Glioma Tissue
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As shown in the flowchart (Fig. S1 a), glioma tissues were minced with scissors, digested with acctuase (SCR005, Sigma), and filtered into a single-cell suspension in a 40-µm cell strainer (352,340, Falcon). After removal of red blood cells using red blood cell lysis buffer (B541001, Sangon Biotech), cell suspension was diluted in magnetic affinity cell sorting (MACS) buffer (130-091-221, Miltenyi Biotec) and incubated with CD11b microbeads (130-093-634, Miltenyi Biotec) for 15 min at 4 ℃. The cell suspension was applied to the MS column (130-042-201, Miltenyi Biotec). CD11b-positive cells were then labelled by a washing column. After pressing the plunger firmly into the column, positive cells were eluted and collected [23 (link)]. Flow cytometry was used to verify the cell purity (Fig. S1 b, c).
5
Isolating Mouse Immune Cells
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Red blood cells from mouse spleens were lysed using Red Blood Cell Lysis buffer (Sangon Biotech, China). OT-I CD8+ T cells from OT-I transgenic mice were purified using the MojoSortTM Mouse CD3 T cell Isolation kit (BioLegend, USA). Harvested tumors were dissociated with 0.2 mg/mL DNase I (Sigma-Aldrich, USA) and 0.05 mg/mL Liberase TL (Roche, Germany) in RPMI for 25 min at 37°C.
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