在生物科研领域，经常需要去除红细胞。去除红细胞的方法有多种，如ACK Lysis Buffer、Tris-氯化铵红细胞裂解液、Gey's Lysis Buffer。Gey's Lysis Buffer是一种从人、鼠或其他哺乳动物等体内的组织样品或血液中裂解并去除无核红细胞的溶液，主要由磷酸盐、葡萄糖、NH4Cl等组成。
Gey's Lysis Buffer经过优化配方，含碳酸盐、Ca2+、Mg2+，在裂解无核红细胞的同时几乎不损伤淋巴细胞 (Lymphocyte)或其它有细胞核的细胞，本裂解液经高压灭菌及过滤除菌，经过Gey's Lysis Buffer处理过的血液或组织细胞样品可以用于后续的细胞培养、细胞融合以及核酸或蛋白的提取及各种常规的分析和检测，尤其适用于去除皮细胞脾细胞中的红细胞。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、胰蛋白酶、胎牛血清 (FCS) 、Hanks平衡盐溶液 (HBSS)
2、低温离心机
操作步骤 (仅供参考)：
(一)组织细胞样本
1、制备细胞悬液: 新鲜组织经过胰蛋白酶或胶原酶等消化处理，通过适当方法制备成细胞悬液，离心弃上清。
2、裂解: 加入5倍细胞沉淀体积的Gey's Lysis Buffer，轻柔吹打混匀，立即冰浴裂解5min。
3、加入100% FCS，4℃ 300g离心10min，弃红色上清；如无低温离心机，本步骤亦可在室温下操作。
4、如果发现红细胞裂解不完全，可以重复上述步骤2和步骤3各一次。
5、洗涤：根据实验要求加入适量HBSS，轻柔混匀重悬沉淀，4℃ 400-500g离心2-3min，弃上清，该离心步骤亦可在室温下操作，所加入HBSS的量一般应大于细胞沉淀体积的5倍以上。
6、如有必要，重复上述步骤5一次，共洗涤1-2次。
7、根据实验需要用适当溶液重悬细胞沉淀，进行计数、培养等后续实验。
(二)血液样本
1、取新鲜抗凝血，400-500g离心5min，弃上清。
2、 裂解: 加入6-10倍细胞沉淀体积的Gey's Lysis Buffer，轻柔吹打混匀，立即冰浴裂解5min (特别提醒：对于鼠的血液，裂解5min已经足够，对于人的外周血，宜延长裂解时间至5-10min，并且裂解过程中轻轻摇动以促进红细胞裂解)。
3、加入100% FCS，4℃ 300g离心10-15min，弃红色上清，如无低温离心机，本步骤亦可在室温下操作。
4、如果发现红细胞裂解不完全，可以重复上述步骤2和步骤3一次。
5、洗涤: 根据实验要求加入适量HBSS，轻柔混匀重悬沉淀，4℃ 400-500g离心2-3min，弃上清，该离心步骤亦可在室温下操作，所加入HBSS的量一般应大于细胞沉淀体积的5倍以上。
6、根据实验需要用适当溶液重悬细胞沉淀，进行计数、培养等后续实验。
注意：对于微量或少量的血液样本，可以不用第1步操作，可直接加入10倍血液体积的Gey's Lysis Buffer进行第2步操作，并在4℃裂解10-15min；对于鼠的血液，裂解15min已经足够；对于人的外周血，宜延长裂解时间至20min，但通常不宜超过20min，并且裂解过程中宜适当摇动以促进红细胞裂解。
注意事项：
1、制备细胞悬液时应根据实验需要，不一定要制备成单细胞悬液。
2、后续试验如果是用于细胞培养，操作过程中应注意无菌操作，尽量在超净工作台内操作。
3、离心步骤尽量在4℃离心机上操作。
5、离心洗涤后，通常极微量的红细胞不会影响后续的检测。
6、如果经过Gey's Lysis Buffer处理后的样品后续用于总RNA的提取，在处理细胞时不必使用DEPC处理的溶液，即无需在该操作中特意去除RNase。
7、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Product Introduction
In the field of biological research, red blood cell removal is frequently required. There are various methods for red blood cell removal, such as ACK Lysis Buffer, Tris-ammonium chloride red blood cell lysis buffer, and Gey's Lysis Buffer. Gey's Lysis Buffer is a solution used to lyse and remove enucleated red blood cells from tissue samples or blood of humans, mice, other mammals, etc. It is mainly composed of phosphate, glucose, NH4Cl, and other ingredients.
The formula of Gey's Lysis Buffer has been optimized, containing carbonate, Ca²⁺, and Mg²⁺. It can lyse enucleated red blood cells while causing almost no damage to lymphocytes or other nucleated cells. This lysis buffer is sterilized by autoclaving and filtration. Blood or tissue cell samples treated with Gey's Lysis Buffer can be used for subsequent experiments including cell culture, cell fusion, nucleic acid or protein extraction, as well as various routine analyses and detections. It is especially suitable for removing red blood cells from skin cells and splenocytes. This reagent is for research use only, not for clinical diagnosis or any other purposes.
Materials to Be Prepared by Users
1. Trypsin, fetal calf serum (FCS), Hanks' balanced salt solution (HBSS)
2. Low-temperature centrifuge
Operating Procedures (for reference only)
(1) For Tissue Cell Samples
1. Prepare cell suspension: Digest fresh tissues with trypsin or collagenase, prepare a cell suspension using an appropriate method, and centrifuge to discard the supernatant.
2. Lysis: Add Gey's Lysis Buffer at a volume 5 times that of the cell pellet, gently pipette to mix well, and immediately place on ice for lysis for 5 minutes.
3. Add 100% FCS, centrifuge at 300g for 10 minutes at 4℃, and discard the red supernatant. This step can also be performed at room temperature if a low-temperature centrifuge is not available.
4. If red blood cell lysis is incomplete, repeat steps 2 and 3 once each.
5. Washing: Add an appropriate amount of HBSS according to experimental requirements, gently mix to resuspend the pellet, centrifuge at 400-500g for 2-3 minutes at 4℃, and discard the supernatant. This centrifugation step can also be performed at room temperature. The volume of HBSS added should generally be more than 5 times that of the cell pellet.
6. If necessary, repeat step 5 once, with a total of 1-2 washing cycles.
7. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed to subsequent experiments such as cell counting and culture.
(2) For Blood Samples
1. Take fresh anticoagulated blood, centrifuge at 400-500g for 5 minutes, and discard the supernatant.
2. Lysis: Add Gey's Lysis Buffer at a volume 6-10 times that of the cell pellet, gently pipette to mix well, and immediately place on ice for lysis for 5 minutes.
Important Note: For mouse blood, 5 minutes of lysis is sufficient. For human peripheral blood, the lysis time should be extended to 5-10 minutes, and the mixture should be gently shaken during lysis to facilitate red blood cell lysis.
3. Add 100% FCS, centrifuge at 300g for 10-15 minutes at 4℃, and discard the red supernatant. This step can also be performed at room temperature if a low-temperature centrifuge is not available.
4. If red blood cell lysis is incomplete, repeat steps 2 and 3 once.
5. Washing: Add an appropriate amount of HBSS according to experimental requirements, gently mix to resuspend the pellet, centrifuge at 400-500g for 2-3 minutes at 4℃, and discard the supernatant. This centrifugation step can also be performed at room temperature. The volume of HBSS added should generally be more than 5 times that of the cell pellet.
6. Resuspend the cell pellet with an appropriate solution according to experimental needs, and proceed to subsequent experiments such as cell counting and culture.
Note: For trace or small-volume blood samples, step 1 can be omitted. Directly add Gey's Lysis Buffer at a volume 10 times that of the blood and proceed to step 2, with lysis performed at 4℃ for 10-15 minutes. For mouse blood, 15 minutes of lysis is sufficient; for human peripheral blood, the lysis time should be extended to 20 minutes, but generally should not exceed 20 minutes. The mixture should be shaken appropriately during lysis to promote red blood cell lysis.
Precautions
1. When preparing the cell suspension, it is not necessary to prepare a single-cell suspension unless required by the experiment.
2. If subsequent experiments involve cell culture, sterile techniques should be strictly followed, and operations should be performed in a clean bench as much as possible.
3. Centrifugation steps should be preferably carried out using a 4℃ centrifuge.
4. After centrifugation and washing, trace amounts of residual red blood cells usually do not affect subsequent detection.
5. If samples treated with Gey's Lysis Buffer are used for total RNA extraction, there is no need to use DEPC-treated solutions during cell processing, i.e., RNase removal is not required in this operation.
6. For your safety and health, wear a lab coat and disposable gloves during operation.
ZJ26F1039668
危险品化学品经营许可证（带存储）