Spleens were harvested from EAMG induced animals and cells were teased from mouse spleens with sterile forceps and passed through a 70 µm filters (BD Falcon) in DMEM. Splenocytes were centrifuged and the pellet was re-suspended in RBC lysis buffer (R&D Systems). Cells were washed twice and re-suspended in complete RPMI culture medium. Splenocytes were frozen in DMEM containing 10% fetal bovine serum and 10% DMSO [33] (link).
Rbc lysis buffer
Manufactured by R&D Systems
Sourced in United States
The RBC lysis buffer is a reagent used to selectively lyse (break down) red blood cells (RBCs) in a sample. It is a key tool in sample preparation for various downstream applications, such as flow cytometry and cell analysis. The buffer acts by disrupting the RBC cell membranes, allowing the release of the cellular contents while leaving other cell types intact. This process helps to enrich the sample for the desired cell populations, facilitating more accurate analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Collagenase 4, by Merck Group (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Merck Group (1 mentions)
Dnase 1 mix, by Roche (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
Rat and mouse serum, by Merck Group (1 mentions)
40 μm cell strainer, by Thermo Fisher Scientific (1 mentions)
Ic fixation buffer, by Thermo Fisher Scientific (1 mentions)
3 protocols using rbc lysis buffer
1
Isolation and Cryopreservation of Murine Splenocytes
2
Joint and Lymph Node Extraction
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Extracted joints were digested in digestion medium containing dispase (2 U/ml; Invitrogen), collagenase IV (20 mg/ml; Sigma-Aldrich), and DNase I mix (50 mg/ml; Roche Applied Science) in complete RPMI medium, followed by red blood cell (RBC) lysis using RBC lysis buffer (R&D Systems, catalog no. WL2000). Cell debris and skin tissues were removed by passing through 40-μm-pore-size cell strainer and spinning down in 35% (vol/vol) Percoll solution in RPMI (Sigma-Aldrich, catalog no. P1644-1L) prior to staining.
The popliteal lymph node (pLN), which is located at the area to the back of the mice knee joint, was retrieved and incubated in 1 ml of digestion medium containing dispase (2 U/ml), collagenase IV (20 mg/ml), and DNase I mix (50 mg/ml) in complete RPMI medium for 30 min at 37°C. Both pLN and digestion medium were mixed well before passing contents through a nylon mesh cloth (70-μm pore size; Sefar). The RBCs were further lysed prior to additional staining procedures.
The popliteal lymph node (pLN), which is located at the area to the back of the mice knee joint, was retrieved and incubated in 1 ml of digestion medium containing dispase (2 U/ml), collagenase IV (20 mg/ml), and DNase I mix (50 mg/ml) in complete RPMI medium for 30 min at 37°C. Both pLN and digestion medium were mixed well before passing contents through a nylon mesh cloth (70-μm pore size; Sefar). The RBCs were further lysed prior to additional staining procedures.
3
Murine Splenic Cell Isolation and Flow Cytometric Analysis
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Spleens of mice were surgically extracted and dissociated in RPMI medium containing 10% FBS (complete RPMI) and passed through a 40 μm cell strainer (Fisherbrand), followed by RBC lysis with RBC lysis buffer (R&D system, Minneapolis, MN, USA). Isolated cells were stained with LIVE/DEAD Aqua (Life Technologies) and then incubated in 100 μL blocking buffer consisting of a mix of 1% rat and mouse serum (Sigma‐Aldrich) in FACS buffer (1% BSA, 2 μm EDTA in PBS). Next, cells were stained with conjugated antibodies for 20 min and fixed in IC fixation buffer (eBioscience, San Diego, CA, USA) for 5 min before acquisition using a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Conjugated antibodies used were CD45 (BD Bioscience), B220 (eBioscience), GL‐7 (BD Biosciences), CD95 (BD Biosciences), CD38 (Biolegend, San Diego, CA, USA), CD73 (eBioscience), CD138 (BD Bioscience) and IgD (BD Bioscience).
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