Flow Cytometry Protocol for mouse heart, bone marrow, spleen, and blood
小鼠心脏、骨髓、脾脏和血液的流式细胞术方案

Notes to keep cell viability: ①keep your samples on ice during all your procedure. ②Keep your cells in equal osmotic pressure solution/buffer, such as DMEM, PBS, FACS buffer, ACK lysis buffer. A drop of water may kill your cells.
保持细胞活力的注意事项：（1）在所有过程中将样品放在冰上。（2）将细胞保持在渗透压相等的溶液/缓冲液中，如DMEM、PBS、FACS、ACK裂解缓冲液。一滴水可能会杀死你的细胞。

Centrifugation: 1200-1600 rpm would be best speed for all the live cells centrifugation. You can do 1200 rpm for 6 minutes or 1600 rpm for 5 minutes if you are analyzing big cells, such as macrophages. You can do 2000 rpm for 5-10 minutes if you are analyzing smaller cells, such as T cells. Gently remove the supernatant without disturbing the pellets.
离心：1200-1600 rpm 是所有活细胞离心的最佳速度。如果您正在分析大细胞，例如巨噬细胞，您可以进行 1200 rpm 6 分钟或 1600 rpm 5 分钟。如果您正在分析较小的细胞，例如 T 细胞，您可以以 2000 rpm 的速度进行 5-10 分钟。轻轻除去上清液，不要干扰沉淀。

Commonly used Reagents for mouse tissue single cells isolation:
小鼠组织单细胞分离常用试剂：

DMEM 1X, PBS, EDTA (0.5 M, pH8, corning REF 46-034-CI, LOT 08819001).
DMEM 1X，PBS，EDTA（0.5 M，pH8，康宁编号 46-034-CI，批次 08819001）。

3 enzymes for heart digestion：①Collagenase is the major enzyme for heart digestion. You can use collagenase 1 (Sigma C0130-1G-125000 units, dilute to 4500 units/mL with DMEM), which is a modest enzyme, need almost 1 hour digestion. You can also choose collagenase IV (Sigma C5138-1G-125000 units, dilute to 4500 units/mL with DMEM), which is a strong enzyme, only takes 30 minutes to digest a whole heart. ② Hyaluronidase aims to digest hyaluronic acid, which is sticky and will block cell strainer or flow machine. We choose Hyaluronidase type 1-s (Sigma H3506-500MG, dilute to 6000 units/mL with DMEM). ③DNAse can be reused, we choose DNAse 1 (Sigma D4527-14mg-28000 units, dilute to 2400 units/mL with DMEM) or DNAse 1 (SKU # 11284932001), Sigma. Equal proportion volume of enzymes will make your digestion setting easier. For example, you can aliquot 1200 uL collagenase 1, 600 uL Hyaluronidase I and 120 uL DNAse I for 4 hearts.
3种心脏消化酶：（1）胶原酶是心脏消化的主要酶。您可以使用胶原酶 1（Sigma C0130-1G-125000 单位，用 DMEM 稀释至 4500 单位/mL），这是一种适度的酶，需要近 1 小时的消化。也可以选择胶原酶IV（Sigma C5138-1G-125000单位，用DMEM稀释至4500单位/mL），这是一种强效酶，只需30分钟即可消化整个心脏。（2）透明质酸酶旨在消化透明质酸，透明质酸具有粘性，会阻塞细胞过滤器或流动机。我们选择透明质酸酶 1-s 型（Sigma H3506-500MG，用 DMEM 稀释至 6000 单位/mL）。（3）DNA酶可重复使用，我们选择DNAse 1（Sigma D4527-14mg-28000单位，用DMEM稀释至2400单位/mL）或DNAse 1（SKU # 11284932001），Sigma。等比例体积的酶将使您的消化设置更容易。例如，您可以将 1200 uL 胶原酶 1、600 uL 透明质酸酶 I 和 120 uL DNAse I 等分用于 4 颗心脏。

建立消化系统:：取15ml离心管，加入DMEM (2.5 mL)、胶原酶1(300µL，最终浓度为450U/mL)、透明质酸酶(75µL，最终浓度为60U/mL)和DNAse1(30µL，最终浓度为60u/mL)

digestion deactivating reagent：Bovine serum albumin and FBS (56℃ heat inactivated for 30 minutes)
消解灭活试剂：牛血清白蛋白和全血细胞计数（56°C加热灭活30分钟）

Kit for intracellular staining (no need for surface staining): Check if all your antibodies are surface marker, if not, you will need a True-Nuclear™ Transcription Factor Buffer Set (biolegend 424401) to do fixation and permeablization staining. Try to avoid intracellular if you can find a surface marker for your analysis.
细胞内染色试剂盒（无需表面染色）：检查您的所有抗体是否都是表面标记物，如果不是，您将需要真核™转录因子缓冲液组（biolegend 424401）进行固定和透化染色。如果可以找到用于分析的表面标记物，请尽量避免细胞内使用。

Red cells elimination buffer: ACK lysis buffer (),
红细胞消除缓冲液：ACK裂解缓冲液（），

PBB solution for deactivating heart digestion: 490 mL PBS + 10 mL FBS + 1g BSA
用于灭活心脏消化的多溴联苯溶液：490 mL PBS + 10 mL FBS + 1g BSA

FACS Buffer：976 mL PBS (1X, without Ca²⁺ and Mg²⁺) + 20mL FBS + 4ml EDTA (0.5M)
FACS 缓冲液：976 mL PBS（1X，不含 Ca ²⁺ 和 Mg ²⁺ ）+ 20mL FBS + 4ml EDTA （0.5M）

Materials for tissue harvest and treatment: Filters, tubes (50 mL, 15 mL, 1.5 mL), 1 ml and 3-10 ml syringes, scissors, and tweezers.
组织采集和处理材料：过滤器、试管（50 mL、15 mL、1.5 mL）、1 ml 和 3-10 ml 注射器、剪刀和镊子。

How to pick flow cytometry antibodies?
如何挑选流式细胞术抗体？

Based on protein expression and dye fluorescence signal, we should choose the stronger colors for low expressed proteins and choose relative lower colors for high expressed proteins. PE, APC, PE/Cy7, APC/Cy7, BV421, BV510, FITC and PercP/Cy5.5 are most common used dyes for flow cytometry antibodies because of their relative low price and strong brightness. PE is the strongest dye (brightness *****), so PE is always used for your targeted antibody or some low expressed proteins. APC and BV421 are the second strongest dye (****). PE/Cy7, APC/Cy7, BV510 are also very strong (***). FITC and PercP/Cy5.5 are weakest of these 8 dyes (**), so these two colors are always used for most high expressed CD45 in injured tissues, or CD11b, Ly6G, CX3CR1, et.al in innate immune responded injured tissues. Actually, even FITC and PercP/Cy5.5 are strong enough for flow cytometry. Since a lot of reporter mice have GFP, we cannot use FITC channel, so PercP/Cy5.5 is always used for CD45.
根据蛋白质表达和染料荧光信号，低表达蛋白应选择较强的颜色，高表达蛋白应选择相对较低的颜色。PE、APC、PE/Cy7、APC/Cy7、BV421、BV510、FITC和PercP/Cy5.5是流式细胞术抗体最常用的染料，因为它们的价格相对较低，亮度高。PE是最强的染料（亮度*****），因此PE始终用于靶向抗体或一些低表达蛋白质。APC 和 BV421 是第二强的染料 （****）。PE/Cy7、APC/Cy7、BV510 也非常强 （***）。FITC 和 PercP/Cy5.5 是这 8 种染料中最弱的 （**），因此这两种颜色始终用于损伤组织中表达最多的 CD45，或先天免疫反应损伤组织中的 CD11b、Ly6G、CX3CR1 et.al。实际上，即使是 FITC 和 PercP/Cy5.5 也足以用于流式细胞术。由于许多报告小鼠具有GFP，因此我们不能使用FITC通道，因此始终使用PercP / Cy5.5用于CD45。

可以使用thermoSpectraViewer判断染料overlap

https://www.thermofisher.cn/order/fluorescence-spectraviewer#!/

The following two graphs are from Wikipedia demonstrating brief haematopoiesis.
以下两张图表来自维基百科，展示了短暂的造血。

Diagram showing the development of different blood cells from haematopoietic stem cell to mature cells.
从造血干细胞到成熟细胞的不同血细胞的发育图。

Hematopoiesis (human) diagram showing the development of different blood cells from haematopoietic stem cell to mature cells in both myeloid and lymphoid lineages.
造血（人类）图显示了不同血细胞从造血干细胞到骨髓和淋巴谱系中成熟细胞的发育。

Cell composition of the adult human heart. Nature. 2020 Dec;588(7838):466-472. doi:
成人心脏的细胞组成。自然界。2020年12月;588(7838):466-472.doi： 10.1038/s41586-020-2797-4.10.1038/s41586-020-2797-4.
成人心脏的细胞组成。自然界。2020年12月;588(7838):466-472.doi： 10.1038/s41586-020-2797-4 .

Abbreviation: macrophage (Mφ), activated macrophage (aMφ), resident macrophage (rMφ), monocyte (Mo), dendritic cell (DC), conventional dendritic cell (cDC), plasmacytoid dendritic cell (pDC), antigen-presenting cell (APC), granulocytes (Gr), peripheral granulocyte (pGr), Osteoclast (Os), neutrophil (NE), natural killer cell (NK), mast cell (Mc), thymocyte (th), T cell (T), naïve T cell (nT), CD4+ T cell (T4), CD8+ T cell (T8), B cell (B), regulatory T cell (Treg), memory T (mT), activated T (aT), bone marrow (BM), lymphoid cell (Lym), myeloid cell (Mye), leukocyte (Leu), platelet (pla), endothelial cell (ed), epithelial cell (ep), fibroblast (fb), erythrocyte (RBC). * means major expressed cells, no * means average expression in different cells or I am not sure. – means same as upper. Efficiency in heart, “not sure” means I never used this antibody, “not sure” means I have used this antibody but need check my data to confirm.115
缩写：巨噬细胞（Mφ）、活化巨噬细胞（aMφ）、驻留巨噬细胞（rMφ）、单核细胞（Mo）、树突状细胞（DC）、常规树突状细胞（cDC）、浆细胞样树突状细胞（pDC）、抗原呈递细胞（APC）、粒细胞（Gr）、外周粒细胞（pGr）、破骨细胞（Os）、中性粒细胞（NE）、自然杀伤细胞（NK）、肥大细胞（Mc）、胸腺细胞（th）、T细胞（T）、幼稚T细胞（nT）、CD4+ T细胞（T4）、CD8+ T细胞（T8）、 B 细胞 （B）、调节性 T 细胞 （Treg）、记忆 T （mT）、活化 T （aT）、骨髓 （BM）、淋巴细胞 （Lym）、髓系细胞 （Mye）、白细胞 （Leu）、血小板 （pla）、内皮细胞 （ed）、上皮细胞 （ep）、成纤维细胞 （fb）、红细胞 （RBC）。* 表示主要表达细胞，否 * 表示不同细胞的平均表达，或者我不确定。– 表示与上部相同。“不确定”表示我从未使用过这种抗体，“不确定”表示我使用过这种抗体，但需要检查我的数据来确认115。

Spleen compensation Staining
脾脏代偿染色

1. Harvest and break the spleen (left side when Belly downward).
1.收割并打破脾脏（腹部朝下时左侧）。

2. Rinse the tissue with 5-10 mL DMEM, filter and transfer to a 15 mL tube.
2. 用 5-10 mL DMEM 冲洗组织，过滤并转移到 15 mL 管中。

3. Spin down to pellet cells, then resuspend the cells with 1 mL ACK lysing buffer.
3. 向下旋转至沉淀细胞，然后用 1 mL ACK 裂解缓冲液重悬细胞。

4. Leave the tube on ice for 10 minutes (vortex every 3 minutes) before adding 10 mL DMEM.
4. 在加入 10 mL DMEM 之前，将试管放在冰上 10 分钟（每 3 分钟涡旋一次）。

5. Filter the cells, spin and resuspend with 1-2 mL FACS buffer.
5. 过滤细胞，用 1-2 mL FACS 缓冲液离心并重悬。

6. Divide the spleen samples into 4-8 tubes (100 µL/tube). Add 0.2-0.5 µL antibody to each single staining tube for compensation and incubate for 10-30 minutes in dark. For most Fortessa, LSR and X20 machines, the following 8 colors panel would be your best choice: ①Percy/Cy5.5-CD45 ②PE-CD3 ③APC-CD4 ④PE/Cy7-CD4 ⑤APC/Cy7-CD8a ⑥ BV421-CD4 ⑦ FITC-CD19 ⑧BV510-CD19. You can set all the 8 colors but only stain your own sample with some of the colors antibodies, like 5-6 antibodies. Or you can just do 5-6 colors compensation for you own samples because compensation is kind of challenging for new leaners. First delete BV510 if you have only 7 antibodies, you can also delete APC/Cy7 if you have only 6 antibodies, and PE/Cy7 if you only have 5 antibodies. That’s because these colors have sort of big overlap with other colors, especially BV510.
6. 将脾脏样品分成 4-8 管 （100 μL/管）。向每个单染色管中加入 0.2-0.5 μL 抗体进行补偿，并在黑暗中孵育 10-30 分钟。对于大多数 Fortessa、LSR 和 X20 机器，以下 8 种颜色面板将是您的最佳选择：（1）Percy/Cy5.5-CD45 （2）PE-CD3 （3）APC-CD4 （4）PE/Cy7-CD4 （5）APC/Cy7-CD8a （6） BV421-CD4 （7） FITC-CD19 （8）BV510-CD19。您可以设置所有 8 种颜色，但只能使用某些颜色的抗体（如 5-6 种抗体）对自己的样品进行染色。或者你可以只为自己的样品做 5-6 种颜色的补偿，因为补偿对新人来说有点挑战性。如果您只有 7 种抗体，首先删除 BV510，如果您只有 6 种抗体，也可以删除 APC/Cy7，如果您只有 5 种抗体，也可以删除 PE/Cy7。这是因为这些颜色与其他颜色有很大的重叠，尤其是BV510。

7. Add 1 mL FACS buffer to wash the antibodies, spin down to pellet the cells.
7. 加入 1 mL FACS 缓冲液洗涤抗体，向下旋转以沉淀细胞。

8. Resuspend the pellets with 200 uL FACS buffer and transfer to the flow tube by pipetting through the flow tube strainer. Now it is ready for flow analysis.
8. 用 200 uL FACS 缓冲液重悬沉淀，并通过流管过滤器移液转移到流管中。现在，它已准备好进行流量分析。

Mice hearts Flow Cytometry Protocol
小鼠心脏流式细胞术方案

A: Samples Preparation
A： 样品制备

Design and check with the antibodies before injection or surgery.
在注射或手术前设计并检查抗体。

Setting up the digestions: Take the 15 mL conical tube, add DMEM (2.5 mL), Collagenase 1 (300 µL, final as 450 units/mL), Hyaluronidase (75 µL, final as 150 units/mL) and DNAse 1 (30 µL, final as 60 units /mL).
设置酶解：取 15 mL 锥形管，加入 DMEM（2.5 mL）、胶原酶 1（300 μL，最终为 450 单位/mL）、透明质酸酶（75 μL，最终为 150 单位/mL）和 DNAse 1（30 μL，最终为 60 单位/mL）。

Sacrifice the mouse, take out the heart and perfuse the hearts with PBS.
牺牲老鼠，取出心脏，用PBS灌注心脏。

Weigh the hearts. Cut the heart into two parts if you need some heart tissue for other experiment, you will need at least half heart for Flow cytometry.
称量心。将心脏切成两部分 如果您需要一些心脏组织进行其他实验，则至少需要一半的心脏进行流式细胞术。

Mince the tissue chunk into fine pieces using a blade or a scissor.
用刀片或剪刀将纸巾块切成细块。

Add the minced tissue into the tube. Mix well with tips. Digest for 1 hour at 37 ℃ with quick shaking (around 200 rpm).
将切碎的组织加入试管中。与小费混合均匀。在37°C下快速振荡（约200rpm）消化1小时。

Add ~5 mL of PBB to the digestion tubes to deactivate the digestion.
向消解管中加入~5 mL多溴联苯以灭活消解。

Pour the mixture of step 7 through a 50 µm wet filter on the 50 mL conical tubes.
将步骤 7 的混合物倒入 50 mL 锥形管上的 50 μm 湿过滤器。

Transfer (pour) the samples back into fresh 15 mL conical tube, spin the samples at 1200 rpm for 6 minutes (4℃).
将样品转移（倒入）回新鲜的 15 mL 锥形管中，以 1200 rpm 的速度旋转样品 6 分钟 （4°C）。

Resuspend the pellet with 1 mL of ACK lysis buffer. Perform RBC lysis at room temp for 5 minutes.
用 1 mL ACK 裂解缓冲液重悬沉淀。在室温下进行红细胞裂解 5 分钟。

Add 9 mL of DMEM or FACS buffer to the sample to wash off ACK buffer.
向样品中加入 9 mL DMEM 或 FACS 缓冲液以洗去 ACK 缓冲液。

Spin again and discard the supernatant.
再次旋转并丢弃上清液。

Add 2 mL FACS buffer, resuspend the pellet and transfer to a 5 mL tube.
加入 2 mL FACS 缓冲液，重悬沉淀并转移到 5 mL 试管中。

Spin again and leave 100-200 µL of FACS Buffer.
再次旋转并留下 100-200 μL FACS 缓冲液。

B: Surface Marker Antibodies Stainings
B：表面标记抗体染色

Stain with the mixture of the surface marker antibodies for 30 minutes at 4℃ in dark.
用表面标记抗体的混合物在 4°C 的黑暗中染色 30 分钟。

Add 2 mL FACS buffer, spin and discard the supernatant.
加入 2 mL FACS 缓冲液，旋转并弃去上清液。

Resuspend in 200 µL FACS buffer Prior to flow cytometric analysis.
在流式细胞术分析之前重悬于200μL FACS缓冲液中。

C: Intracellular Marker Antibodies Stain (if needed)
C：细胞内标志物抗体染色（如果需要）

Wash cells 1 time with 2 mL FACS buffer and pellet by centrifugation.
用 2 mL FACS 缓冲液洗涤细胞 1 次，并通过离心沉淀。

Dilute True-Nuclear™ 4X Fix Concentrate with True-Nuclear^TM Fix Dilute to get the 1X fix buffer. Resuspend pellet with 1 mL 1X Fix buffer to each tube. Incubate at room temperature in the dark for 45-60 minutes
用 True-Nuclear ^TM Fix Dilute 稀释 True-Nuclear™ 4X Fix 浓缩液以获得 1X 修复缓冲液。将含有 1 mL 1X Fix 缓冲液的沉淀重悬到每个试管中。在室温下在黑暗中孵育 45-60 分钟.

Dilute True-Nuclear™ 10X Perm Buffer with distilled water to get 1X Perm Buffer. Don’t wash mixture of step 19, add 2 mL of 1X Perm Buffer to each tube. Spin and discard the supernatant.
用蒸馏水稀释 True-Nuclear™ 10X Perm 缓冲液，得到 1X Perm 缓冲液。不要洗涤步骤 19 的混合物，向每个试管中加入 2 mL 1X Perm 缓冲液。旋转并弃去上清液。

Add 2 mL of the True-Nuclear™ 1X Perm Buffer, spin and discard the supernatant.
加入 2 mL True-Nuclear™ 1X Perm 缓冲液，旋转并弃去上清液。

Resuspend pellet with 100-150 µL True-Nuclear™ 1X Perm Buffer and add antibody. Incubate cells in the dark at room temperature for at least 30 minutes.
用 100-150 μL True-Nuclear™ 1X Perm 缓冲液重悬沉淀并加入抗体。在室温下在黑暗中孵育细胞至少 30 分钟。

Add 2 mL True-Nuclear™ 1X Perm Buffer, spin and discard the supernatant.
加入 2 mL True-Nuclear 1X™ Perm 缓冲液，旋转并弃去上清液。

Wash cells 1 time with 2 mL FACS buffer in tubes, spin and discard the supernatant.
用试管中的 2 mL FACS 缓冲液洗涤细胞 1 次，旋转并弃去上清液。

Resuspend in 200 µL FACS buffer Prior to flow cytometric analysis.
在流式细胞术分析之前重悬于200μL FACS缓冲液中。

D: Common Flow Machine Operation
D：共流机器操作

Login in, user name: GuoshuaiFeng; password: Fgs-1987.
登录，用户名：GuoshuaiFeng;密码：Fgs-1987。

New experiment—click blank experiment.
新实验 - 单击空白实验。

Set parameters: view—inspector—cytometer setting—parameters—click SSC/FSC, chose A, H and W—delete unwanted parameter
设置参数：查看—检测器—细胞仪设置—参数—单击SSC/FSC，选择A、H和W—删除不需要的参数

Create compensation: experiment—compensation setup—create compensation control—name different colors.
创建补偿：实验—补偿设置—创建补偿控制—命名不同的颜色。

Check the stain: Click compensation control, and run every color of spleen sample. Separate every color by changing voltage. Make sure unstained compensation all negative, each color compensation positive but other color negative.
检查污渍：点击补偿控制，运行各色脾样。通过改变电压来分离每种颜色。确保未染色的补偿都是负的，每种颜色的补偿都是正的，但其他颜色的补偿是负的。

Run unstained sample, set events to record 5000 and display 5000. Record it and apply to all compensation.
运行未染色的样品，将事件设置为记录 5000 并显示 5000。记录下来并适用于所有赔偿。

Run every color and calculate compensation.
运行每种颜色并计算补偿。

Run samples and change the voltage of FSC and SSC to make sure all the cells in the scope. Record 0.2-0.5 million CD45+ cells and save results.
运行样品并更改 FSC 和 SSC 的电压，以确保所有电池都在示波器中。记录 0.2-50 万个 CD45+ 细胞并保存结果。

Our Rosa26-Td^tomato is bright, more similar with PE/Texas Red than PE channel, so we will use PE/Texas Red channel. Use spleen of CSF1R^icreRosa26-Td^tomato mouse or Flt3^creRosa26-Td^tomato mouse which is constant Td^tomato for compensation and set at 10⁴. Use PE for compensation and check sample before recording when we don’t have CSF1R^icreRosa26-Td^tomato mouse or Flt3^creRosa26-Td^tomato mouse.
我们的 Rosa26-Td ^tomato 很亮，与 PE/Texas Red 比 PE 通道更相似，因此我们将使用 PE/Texas Red 通道。使用CSF1R ^icre Rosa26-Td ^tomato 小鼠或Flt3 ^cre Rosa26-Td ^tomato 小鼠的脾脏，其Td ^tomato 恒定用于补偿并设置为10 ⁴ 。当我们没有 CSF1R ^icre Rosa26-Td ^tomato 小鼠或 Flt3 ^cre Rosa26-Td ^tomato 小鼠时，请使用 PE 进行补偿并在记录前检查样品。

Blood lymphoid
血淋巴

Drop the blood into a 15 mL conical tube containing 8 mL ACK lysing buffer and 750 uL EDTA.
将血液滴入含有 8 mL ACK 裂解缓冲液和 750 uL EDTA 的 15 mL 锥形管中。

Leave the tube on ice for 15 minutes to kill the red blood cells, vortex every 5 minutes.
将试管放在冰上 15 分钟以杀死红细胞，每 5 分钟涡旋一次。

Spin down the cells and resuspend the pellet with 4 mL ACK lysing buffer (no EDTA), put the tube on ice for 5-10 minutes.
旋转细胞并用 4 mL ACK 裂解缓冲液（无 EDTA）重悬沉淀，将试管放在冰上 5-10 分钟。

Spin down the cells and resuspend the pellet with 1 mL ACK lysing buffer (no EDTA), transfer to a 5 mL tube. Put the tube on ice for another 5 minutes.
旋转细胞并用 1 mL ACK 裂解缓冲液（无 EDTA）重悬沉淀，转移到 5 mL 管中。将试管放在冰上再放 5 分钟。

Add 4 mL FACS buffer to wash the cells, spin down and resuspend the pellet with 0.2 mL FACS buffer, and then add antibodies for 20 minutes staining.
加入 4 mL FACS 缓冲液洗涤细胞，旋转并用 0.2 mL FACS 缓冲液重悬沉淀，然后加入抗体染色 20 分钟。

Add 5 mL FACS buffer for another wash
加入 5 mL FACS 缓冲液进行另一次洗涤

Spin down and resuspend with 0.2-0.4 mL FACS buffer for analysis or sorting.
用 0.2-0.4 mL FACS 缓冲液旋转并重悬，用于分析或分选。

Note: there are more lymphoid cell than myeloid cells in blood.
注意：血液中的淋巴细胞多于髓系细胞。

Bone marrow
骨髓

Break the bone marrow with 5 mL or 10 mL pipette.
用 5 mL 或 10 mL 移液管破坏骨髓。

Spin and resuspend the pellet with PBB buffer or HBSS buffer (HBSS+10%FBS) or FACS buffer.
用PBB缓冲液或HBSS缓冲液（HBSS + 10%FBS）或FACS缓冲液旋转并重悬沉淀。

Spin again and leave 100 µL HBSS buffer to resuspend the pellet before staining.
再次旋转并留下 100 μL HBSS 缓冲液以在染色前重悬沉淀。