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。
biolegend anti-mouse FC antibodies for myeloid cells | ||||
antibody | Other names | catlog | Marker for? | Efficiency in heart |
PE-MertK | Mer | 151505 | Mφ*, Mo, DC | not sure |
PE-F4/80 | EMR1 | 123109 | Mφ*, Mo, DC | not sure |
PE-CD64 | FcγRI, FcRI | 139303 | Mφ*, Mo, DC, Mc | excellent |
PE-Ly6G | Lymphocyte antigen 6 complex, locus G | 127607 | BM, pGr | excellent |
PE-CD11c | αX integrin, Itgax | 117307 | DC, NK, Lym | great |
PE-CD103 | Integrin αE chain, Itgae | 121405 | DC*, T, Lym | great |
PE-CD169 | Sialic acid binding Ig-like lectin 1 (Siglec-1) | 142403 | Resident BM Mφ, Gr, Mo, NK, B, T | not sure |
PE-CD172α | SIRPα | 144011 | Mφ*, Mo, Mye, neuron | great |
PE-XCR1 | GPR5, CCXCR1 | 148203 | T8+ cDC | works |
PE-CCR2 | CD192 | 150609 | Mφ, Mo, basophils | excellent |
APC-CD64 | - | 119305 | - | excellent |
APC-F4/80 | - | 123115 | - | not sure |
APC-CD115 | CSF-1R, M-CSFR | 135509 | Mφ, Mo, pDC, cDC, | fail |
APC-CD24 | Heat Stable Antigen (HSA), Nectadrin | 138505 | DC, Gr, Mo, ep, Lym, DC, neuron, | works |
PE/Cy7-CD64 | - | 139313 | - | excellent |
PE/Cy7-CX3CR1 | Chemokine (C-X3-C motif) receptor 1, Fractalkine receptor | 149047 | rMφ, aMφ, Mo, DC, NK, mT, mc, | not sure |
PE/Cy7-Ly6G | - | 127617 | - | not sure |
PE/Cy7-CD45.1 | - | 110729 | - | not sure |
PE/Cy7-CD115 | - | 135523 | - | fail |
PE/Cy7-CCR2 | CD192 | 150611 | Mφ, Mo, basophils | excellent |
APC/Cy7-CX3CR1 | - | 149047 | - | great |
APC/Cy7-F4/80 | - | 123117 | - | not sure |
APC/Cy7-Ly6G | - | 127623 | - | not sure |
BV421-CX3CR1 | - | 149023 | - | great |
BV421-CCR2 | - | 150605 | - | excellent |
BV421-Ly6G | - | 127627 | - | not sure |
BV421-MHCII | MHC class II | 107631 | APC, B, T cells from H-2b,d,q,r bearing mice | excellent |
FITC-CD64 | - | 139315 | - | not sure |
FITC-F4/80 | - | 123108 | - | not sure |
FITC-Ly6G | - | 127606 | - | not sure |
FITC-CD103 | - | 121419 | - | not sure |
biolegend anti-mouse FC antibodies for lymphoid cells | ||||
antibody | Other names | catlog | Marker for? | Efficiency in heart |
PE-CD3 | T cell antigen receptor complex, T3 | 100205 | T cells, NK-T cells, thymocytes during T cell differentiation | works, but not great |
PE-CD19 | B4 | 115507 | all pro-B to mature B cells, follicular dendritic cells | not sure |
PE-CD44 | Hermes, Pgp1, H-CAM, or HUTCH | 139303 | all leukocytes, endothelial cells, hepatocytes, and mesenchymal cells. valuable marker for memory cell subsets | great |
PE-CD62L | L-selectin, LECAM-1, Ly-22, LAM-1, and MEL-14 | 104407 | majority of B, nT, mT, Mo, Grs, most th, and a subset of NK cells. | great |
PE-CD69 | Very Early Activation Antigen (VEA), AIM, EA1, MLR3, gp34/28 | 127607 | rapidly induced on activated T and B cells, neutrophils, and NK cell. a subset of thymocytes and platelets | great |
PE-CD25 | low affinity IL-2Rα, Ly-43, p55, or Tac | 101903 | aT, aB, th, pre-B, Treg | great |
PE-FOXP3 | Integrin αE chain, Itgae | 126403 | Forkhead box protein P3, Scurfin, JM2, or IPEX | great |
APC-CD3 | - | 100235 | - | not sure |
APC-CD4 | - | 100515 | - | great |
APC-CD69 | - | 104513 | - | not sure |
APC-CD44 | - | 103212 | - | not sure |
PE/Cy7-CD3 | - | 100220 | - | not sure |
PE/Cy7-CD4 | - | 100422 | - | great |
PE/Cy7-CD8a | yt-2, Ly-2, or T8 | 100721 | γ/δ TCR-bearing T, NK, intestinal intraepithelial Lym, and lymphoid DC | excellent |
APC/Cy7-CD4 | - | 100413 | - | great |
APC/Cy7-CD8a | - | 100713 | - | great |
BV421-CD3 | - | 100227 | - | not sure |
BV421-CD3e | - | 100335 | - | not sure |
BV421-CD4 | - | 100543 | - | not sure |
BV421-CD8a | - | 100737 | - | not sure |
BV421-CD19 | - | 115537 | - | great |
BV421-CD62L | - | 104435 | - | not sure |
BV510-CD3 | - | 100233 | - | works |
BV510-CD4 | - | 100533 | - | not sure |
BV510-CD19 | - | 139315 | - | not sure |
BV510-NK1.1 | CD161b/c, Ly-55 | 108737 | NK cells and NK-T | not sure |
FITC-CD4 | - | 100406 | - | not sure |
biolegend anti-mouse FC antibodies for universal leukocytes | ||||
antibody | Other names | catlog | Marker for? | Efficiency in heart |
PercP/Cy5.5-CD45 | leukocyte common antigen (LCA), T200, or Ly-5 | 103132 | all hematopoietic cells except mature erythrocytes and platelets. | Excellent in all tissues |
BV510-CD45 | - | 103137 | - | not sure |
PE-CD45.1 | Ly-5.1 | 110708 | all hematopoietic cells except mature erythrocytes and platelets in Ly5.1 bearing mouse strains | not sure |
PE/Cy7-CD11b | αM integrin, Itgam | 101215 | Gr, Mφ, Mo, DC, NK, T, B | excellent |
BV421-CD11b | - | 101235 | - | not sure |
FITC-CD11b | - | 101205 | - | excellent |
PE-Ly6C | Lymphocyte antigen 6 complex, locus C | 128007 | Mφ*, Mo, ed, mT8 | excellent |
APC-Ly6C | - | 128016 | - | great |
APC/Cy7-Ly6C | - | 128025 | - | not sure |
BV510-Ly6C | - | 128033 | - | not sure |
FITC-Ly6C | - | 128006 | - | not sure |
biolegend anti-mouse FC antibodies for non-leukocytes in heart (pending) | ||||
antibody | Other names | catlog | Marker for? | Efficiency in heart |
APC-CD31 | platelet endothelial cell adhesion molecule (PECAM-1), EndoCAM | 103132 | ed*, pla, Gr, Mφ, Mo, DC, T, B | not sure |
FITC-CD31 | - | 102405 | - | not sure |
biolegend anti-mouse FC antibodies for cytokines and receptors (pending) | ||||
antibody | Other names | catlog | Marker for? | Efficiency in heart |
PE-CD121a | IL-1 Receptor type I, IL-1RI, p80 | 113505 | T, th, DC, fb, vascular ed, ep, neural cells | not sure |
APC-CD121a | - | 113509 | - | not sure |
biolegend anti-mouse FC antibodies for non-leukocytes & leukocytes | ||||
antibody | Other names | catlog | Marker for? | Efficiency in heart |
PE-Ly6C | Lymphocyte antigen 6 complex, locus C | 128007 | Mφ*, Mo, ed, mT8 | excellent |
APC-Ly6C | - | 128016 | - | great |
APC/Cy7-Ly6C | - | 128025 | - | not sure |
BV510-Ly6C | - | 128033 | - | not sure |
FITC-Ly6C | - | 128006 | - | not sure |