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Stained samples were incubated with 1× FACS Lyse Solution (Beckton Dickinson) for 10 min and washed twice.
After incubation for 20 min at room temperature in the dark, erythrocytes were lysed with 2 ml fixative-free High-Yield Lyse solution (Life Technologies).
Digested tissue was passed through 70-μm and 40-μm nylon filters, and erythrocytes were lysed in Pharm Lyse solution (BD Biosciences).
Briefly, the red blood cells were lysed for 20 minutes using BD lyse solution (Becton Dickinson Biosciences, San Jose California, USA).
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After two washes with PBS/0.2% BSA and one wash in PBS cells were fixed in 50 µL of BD FACS lysing solution for 15 min at 4 °C.
Fixation and permeabilization (FIX&PERM) solutions A and B (Biozol, Eching, Germany) were used for cytoplasmic anti-κ and anti-λ staining, and BD FACSTM Lysing Solution was used to lyse red blood cells (BD, Heidelberg, Germany).
After staining, red blood cells were lysed with BD FACS Lysing Solution (Becton Dickinson Biosciences, cat. no 349202) and re-suspended to phosphate-buffered saline (PBS) with 2 mM EDTA.
Erythrocytes were lysed using a lysing solution (lysing buffer; BD Pharm Lyse™ BD Biosciencess, Japan) to segregate leukocytes.
This step helps to lyse the cells using lysing solution with detergent.
Presence of high salt in lysing solution helps to remove cell membranes, bulk of proteins, cytoplasm and nucleoplasm.
After washing, the red blood cells were ruptured with 2 mL lysis solution (FACS lysing solution, BD Bioscience).
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